A general unknown screening procedure for drugs and toxic compounds in serum using liquid chromatography-electrospray-single quadrupole mass spectrometry.

A complete general unknown screening procedure was developed using liquid chromatography-mass spectrometry (LC-MS), a coupling that can increase the range of compounds amenable to MS. Sample preparation was by solid-phase extraction on a mixed-mode support in parallel with serum deproteination in order to recover the most hydrophilic compounds. Chromatography employed a reversed-phase narrow-bore column (150 x 1-mm i.d.) and a 50-min gradient elution at low flow-rate (50 microL/min), compatible with the electrospray source used without splitting nor heating. The single quadrupole LC-MS instrument used was operated in the 100 to 1100 mu mass range in both the positive and negative modes, with two different, alternated collision-induced dissociation voltages in the source, in order to obtain the molecular or pseudo-molecular ions as well as fragments for the compounds analyzed. The addition of spectra obtained at low and high fragmentation voltages gave reconstructed spectra for each polarity, representing library entries. Finally, a program was created in order to detect the peaks of interest in the chromatographic noise using a very efficient signal processing algorithm, compute their relative retention time with respect to the internal standard (glafenine), draw their reconstructed spectra, search them in the libraries, and edit a report.

Sensitive determination of vinorelbine and its metabolites in human serum using liquid chromatography-electrospray mass spectrometry.

A liquid chromatography-electrospray mass spectrometry method was developed for the quantitation of vinorelbine (VNB) and two metabolites, vinorelbine N-oxide (VNO) and deacetyl vinorelbine (DAV) in human serum. The limits of quantitation (LOQ) reached 0.5 ng/ml for both VNB and VNO and 1 ng/ml for DAV. The method was proved linear in the range of LOQs up to 1000 ng/ml, and extraction recovery was 80% on average for the three compounds. It was applied to the pharmacokinetic monitoring of vinorelbine and, for the first time, to the detection of VNO in the serum of patients suffering from non-small-cell lung cancer.

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 microm (150 x 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 microl/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 > or = 0.992) and 200 ng/ml for the active metabolites (r > or = 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.

Fatal intoxication following self-administration of a massive dose of buprenorphine.

Several drug packages, including Subutex (high-dose buprenorphine, as sublingual tablets) boxes, were found near the corpse of a 25-year-old male drug addict, who apparently had committed suicide. The autopsy revealed a fatal respiratory depression. The toxicological investigations concluded that death resulted from massive burpienorphine intoxication. The determination of buprenorphine (BU) and norbuprenorphine (NBU) in all biological specimens was performed by liquid chromatography-electrospray mass spectrometry (LC-ES-MS) after hydrolysis (for solid tissues), deproteinization of the matrices, and solid-phase extraction of the compounds. Exceptionally high concentrations of BU and NBU were found in blood (3.3 and 0.4 mg/L, respectively), urine (3.4 and 0.6 mg/L), bile (2035 and 536 mg/L and brain (6.4 a nd 3.9 microg/g). The high concentration of BU (899 mg/L) and the absence of NBU in gastric liquid suggested oral intake. High concentrations of amino-7-flunitra/epam, the main metabolite of flunitra/epam, were also found in blood, urine and gastric liquid. This benzodiazepine may have been a co-factor in the toxic effects of BU.

Sensitive and specific determination of midazolam and 1-hydroxymidazolam in human serum by liquid chromatography-electrospray mass spectrometry.

A liquid chromatographic-mass spectrometric technique was designed for the determination of the anaesthetic benzodiazepine midazolam (MID) and its active metabolite 1-hydroxymidazolam (1-OHM), with the aim of conducting pharmacokinetic/pharmacodynamic studies. MID and 1-OHM were extracted from alkalinised (pH 9.5) spiked and clinical serum samples using a single step, liquid-liquid extraction procedure with diethyl ether-2-propanol (98:2, v/v). The chromatographic separation was performed on a Nucleosil C18, 5 microm (150x1 mm I.D.) column, using a gradient of acetonitrile in 5 mM ammonium formate, pH 3.0 as the mobile phase, delivered at a flow-rate of 50 microl/min. The compounds were ionised in the ionspray source of an atmospheric pressure mass spectrometer, fragmented by in-source collisions and the pseudomolecular and fragment ions detected in the selected ion monitoring mode. The recovery was between 79 and 87% for MID, between 83 and 87% for 1-OHM and 81.5% for methylclonazepam. The limit of detection was 0.2 microg/l for MID and 0.5 microg/l for 1-OHM, the limit of quantitation (LOQ) was 0.5 microg/l for both. Linearity was verified from these LOQs up to 2000 microg/l and the method was found accurate and precise over this range. It was successfully applied to a preliminary study to establish the concentration versus time curve of MID and 1-OHM in a patient administered midazolam by continuous infusion.

Sensitive determination of irinotecan (CPT-11) and its active metabolite SN-38 in human serum using liquid chromatography-electrospray mass spectrometry.

A couple of sensitive and accurate liquid chromatography-electrospray mass spectrometry (LC- S-MS) methods for the determination of the total forms of irinotecan and its active metabolite SN-38 in human serum, using the same chromatographic and detection conditions, is presented. Both used camptothecin as internal standard (I.S.). The sample pretreatment for irinotecan involved a simple protein precipitation with acetonitrile, whereas a liquid-liquid extraction was necessary for SN-38. A Symmetry C18, 3.5 microm (150 x 1 mm I.D.) reversed-phase column was used for the chromatographic separation, together with a gradient elution of acetonitrile in 5 mM ammonium formate buffer (pH 3) as mobile phase. After ionisation in the pneumatically-assisted electrospray source and in-source collision induced dissociation, acquisition was performed in the selected ion monitoring mode. Recoveries were 69 and 47% on average, detection limits 2.5 and 0.25 ng/ml and quantitation limits 10 and 0.5 ng/ml for irinotecan and SN-38, respectively. Reproducibility was good and the method was linear from limits of quantitation up to 10,000 ng/ml for irinotecan, and up to 100 ng/ml for SN-38. This sensitive and highly specific method is suitable both for pharmacokinetic studies and routine therapeutic drug monitoring.

Determination of LSD and N-demethyl-LSD in urine by liquid chromatography coupled to electrospray ionization mass spectrometry.

A sensitive and highly specific method for the determination of LSD and N-demethyl-LSD in urine, using combined liquid chromatography and mass spectrometry (LC-MS) with electrospray ionization, has been developed. Extrelut-3 extraction cartridges were used for a basic sample clean-up. Elution was obtained by toluene-diethyl ether (60:40, v/v). A Nucleosil C18 (150 X 1 mm I.D.) reversed-phase column was used for the chromatographic separation, together with a mixture of 2 mM ammonium formate buffer (pH 3) and acetonitrile (70:30, v/v) as mobile phase. Recoveries were 93 and 80%, detection limits 0.025 and 0.035 ng/ml for LSD and N-demethyl-LSD, respectively. Intra-assay precision, studied at four concentrations, was better than 9% at the ng/ml range and better than 14% at 0.10 ng/ml for both compounds. Limits of quantitation were 0.05 and 0.10 ng/ml for LSD and N-demethyl-LSD, respectively. Reproducibility was good and linearity excellent for LSD in the range from 0.05 to 20 ng/ml (r>0.9999, n=7).

Determination of buprenorphine and norbuprenorphine in whole blood by liquid chromatography-mass spectrometry.

A sensitive and highly specific method for the determination of buprenorphine and norbuprenorphine in postmortem and hemolyzed whole blood using combined liquid chromatography and mass spectrometry (LC-MS) with electrospray ionization was developed. After enzymatic hydrolysis and deproteinization with acetonitrile, Extrelut-3 cartridges were used for a preliminary basic sample cleanup. Elution by toluene-ether (50:50, v/v) was followed by an acid wash with 0.05M H3PO4 and a basic re-extraction into ether. A Nucleosil C18 (150 x 1-mm i.d.) reversed-phase column was used for the chromatographic separation together with a mixture of 2mM ammonium formate buffer (pH 3) and acetonitrile (55:45, v/v) as the mobile phase. Recoveries were between 56 and 60%, and detection limits were 0.05 ng/mL for both analytes. The coefficient of variation for repeatability was lower than 4%. Limits of quantitation were 0.1 ng/mL for buprenorphine and norbuprenorphine. Reproducibility was good, and linearity was excellent in the range of 0.1 to 100 ng/mL (r > 0.9999, n = 7).

Determination of haloperidol and its reduced metabolite in human plasma by liquid chromatography-mass spectrometry with electrospray ionization.

A sensitive and accurate liquid chromatographic-electrospray mass spectrometric (LC-ES-MS) method for the determination of haloperidol (H) and reduced haloperidol (RH) in human plasma is presented, using chlorohaloperidol as the internal standard. A 2-ml volume of plasma was subjected to basic (NaOH) extraction, acid (HCl) back-extraction, acid wash and basic (NaOH) re-extraction. The extraction solvent was hexane-isoamyl alcohol (99:1, v/v) for the whole procedure. A Nucleosil C18 column (150 x 1 mm) was used for high-performance liquid chromatography, together with 2 mM HCOONH4-acetonitrile (55:45, v/v; pH 3.0) as the mobile phase. For each drug, four characteristic ions were monitored. Linearity was assessed in the ranges 0.1-50 and 0.25-50 ng/ml for H and RH, respectively. Recoveries were 58 and 70% and detection limits were 0.075 and 0.100 ng/ml for H and RH, respectively. Correlation coefficients were better than 0.999 for both compounds. R.S.D.s for repeatability and reproducibility at 0.25 ng/ml were 11.1 and 8.5% for H and 9.4 and 11.2% for RH, respectively. One of the main advantages of (LC-ES-MS) over other detection systems is the increase in selectivity obtained by monitoring three ions of confirmation for each of the drugs.

Rapid assay of N-acetyl-beta-D-glucosaminidase isoenzymes in urine by ion-exchange chromatography.

We describe a new method for separating and measuring urinary N-acetyl-beta-D-glucosaminidase isoenzymes by "high-performance" liquid chromatography. Isoenzymes are eluted from the anion-exchange column with a one-step linear gradient of NaCl solution. For continuous post-column quantification of the activities of these isoenzymes, we use an on-line post-column reactor and 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide as substrate; the methylumbelliferone formed in this reaction is quantified fluorimetrically. We discuss the effects of varying different components of the assay: NaCl concentration, the pH of the mobile phase and of the reaction reagent, substrate concentration, incubation temperature, and the geometry of the post-column reactor.

Characterization of the esterases of the mitochondrial fraction of guinea pig cortical renal cells.

The esterase activity of the mitochondrial fraction from cortical renal cells was studied in guinea pigs aged 15, 21, 30, and 120 days. The rate of hydrolysis of beta-naphthyl acetate was measured by incubating aliquots of mitochondrial preparations with physostigmine, diisopropylfluorophosphate, HgCl2, and p-hydroxymercuribenzoate. Enzyme activity was mainly due to the heterogeneous aliesterase group: some aliesterases were sensitive to physostigmine, others to organophosphorus compounds and/or to HgCl2; a C-esterase stimulated by organomercurials and similar to that described by Bergmann et al. in hog kidney was detected (F Bergmann, R Segal, S Rimon. Biochem J 67:481-486, 1957); arylesterase activity was very weak. Acetylthiocholine used as substrate showed there was no cholinesterase activity in the mitochondrial fraction.