Evaluation of automated single mass spectrometry to tandem mass spectrometry function switching for comprehensive drug profiling analysis using a quadrupole time-of-flight mass spectrometer.

A liquid chromatographic mass spectrometric strategy for systematic toxicological analysis (STA) is presented using the automatic 'on-the-fly' single mass spectrometry mode to tandem mass spectrometry mode (MS to MS/MS) switching abilities of a quadrupole time-of-flight (Q-TOF) instrument. During the chromatographic run, the quadrupole is initially set to transmit all masses until (an) ion(s) reaches a certain set threshold. Thereupon, the quadrupole automatically switches to the MS/MS mode, selecting the ion(s), which are subsequently fragmented in the high-efficiency hexapole collision cell, thus generating product ions that are further mass analyzed by the TOF. By limiting the TOF spectral accumulation time in the MS/MS mode to a statistically acceptable minimum, the quadrupole almost instantly switches back to the MS mode. Qualitative information, comprising the complementary MS ([M + H](+) ion mass) and MS/MS (informative product ion profile) data, as well as quantitative information obtained by integration of the MS extracted ion chromatogram(s), can be obtained in one single acquisition. Optimization of the automatic switching parameters, such as threshold, TOF spectral accumulation time, detection window and collision energy, was carried out by injection of a mix of 17 common drugs which were not necessarily baseline separated in the chromatographic system used. Indeed, the complete separation of the drugs is not deemed necessary since up to 8 different ions can 'simultaneously' be selected for MS/MS if they reach the preset criteria. In addition, the quantitative performance of the method was defined. In a second phase, the developed method was field-tested. To that end, the resulting data from extracts of urine samples were compared with and found to be in close concordance with those obtained by a standard toxicological analysis. This innovative approach clearly holds the potential for a substantial advance in the introduction of LC/MS in STA.

Blood-brain barrier equilibration of codeine in rats studied with microdialysis.

PURPOSE: The purpose of the study was to investigate the distribution of codeine across the blood-brain barrier (BBB) in rats by microdialysis (MD). METHODS: Rats were administered intravenous infusion of codeine in doses of (1) 10 mg/kg, (2) 20 mg/kg for 10 min, and (3) an exponential infusion for 2 h aiming at a plasma concentration of 2500 ng/ml, in a crossover design (n = 6). Microdialysis was used to determine codeine unbound concentrations in blood and brain extracellular fluid (ECF). Total brain tissue and plasma concentrations were also determined. Nalorphine was used as a calibrator for measurement of in vivo recovery. RESULTS: Relative recovery and retrodialysis loss of codeine and nalorphine were similar both in vitro and in vivo. Codeine was rapidly transported into the brain ECF with identical influx and efflux clearance across the BBB. The AUC ratios of brain to blood were 0.99 +/- 0.25 and 0.95 +/- 0.16 for Dose 1 and 2, respectively. The Css ratio of brain to blood was 1.06 +/- 0.12 for the exponential infusion. The half-lives were 25 +/- 4 min, 22 +/- 2 min in blood and 27 +/- 5 min, 25 +/- 5 min in brain for Dose 1 and Dose 2, respectively. Total brain tissue concentrations were 3.6 +/- 1.2-fold higher than the unbound concentrations in brain. Codeine was demethylated to morphine with an unbound AUCblood,morphine/AUCblood,codeine ratio of 7.7 +/- 5.1% in blood. No morphine was detected in brain MD, but total concentrations were possible to measure. CONCLUSIONS: Codeine rapidly reached a distributional equilibrium with equal unbound concentrations in blood and brain. The brain transport of codeine did not show any dose-dependency.

Storage temperature effect on the stability of morphine and codeine in urine.

Urine samples collected from one laboratory volunteer and five alleged heroin addicts are prepared (without preservatives) in 5-mL aliquots in glass culture tubes and stored at room, refrigerator, and freezer temperatures. Total morphine, total codeine, free morphine, and free codeine in these samples are analyzed at 30-day intervals for an 11-month period. Total morphine and total codeine concentration decreases are observed for all specimens in all storage conditions. For samples stored in the refrigerator and freezer, similar concentration decrease patterns are observed for total morphine and total codeine, and the decreases range from approximately 10 to 40%. The concentrations of free morphine and free codeine show slight but steady increases. For samples stored at room temperature, large decreases of total morphine are observed for three out of 10 specimens, and total codeine and total morphine concentrations (in seven other specimens) show a decrease pattern similar to that observed for the freezer and refrigerator storage conditions. Three concentration change patterns are observed for free morphine: The type I pattern follows the same decrease pattern observed for freezer and refrigerator storage conditions; the type II pattern shows free morphine increases (after 30-90 days of storage) that remain relatively high for the entire 11-month period; and the type III pattern shows initial increases, followed by gradual decreases to levels that are comparable with the specimens' respective initial concentrations. Free codeine concentrations show slight and steady increases for the entire 11-month period in all specimens.

Rapid and highly selective GC/MS/MS detection of heroin and its metabolites in hair.

A direct treatment of methanol-washed hair with a silylating solution is proposed to extract heroin, O-6-monoacetylmorphine, morphine, acetylcodeine, and codeine, obtaining the simultaneous derivatization of the hydroxylated metabolites and reducing potential sample contamination. Analysis is performed by capillary gas chromatography-tandem mass spectrometry (GC/MS/MS) using multiple selected reaction monitoring. Owing to the selectivity and sensitivity of the GC/MS/MS analysis, and to the extremely simple treatment of the sample, the method fulfils the requirements of both clinical and forensic diagnosis of heroin use.

Comparison of derivatives for determination of codeine and morphine by gas chromatography/mass spectrometry.

Pentafluoropropionic anhydride (PFPA) and heptafluorobutyric anhydride (HFBA) derivatives of morphine and codeine demonstrated poor spectra due to low abundances of secondary and tertiary ions. Trifluoroacetamide (MBTFA) has been a widely used derivative; however, the internal standard, nalorphine, displayed very poor stability and this resulted in split peaks by gas chromatography making MBTFA unsuitable for quantitative methods. Quantitation of codeine and morphine using bis-trimethylsilyltrifluoroacetamide (BSTFA/1%TMS) revealed a significant gradual decrease (p less than 0.05) of peak area ratio (PAR) of codeine and morphine compared to the internal standard using selected ion monitoring (SIM). The acetic anhydride derivative showed no significant differences in the peak area ratios for codeine/IS over a period of 24 hours, although the coefficient of variation (CV) was higher for the acetyl derivative than for the TMS derivative of codeine. There was a significant difference associated with the acetyl derivative of morphine at 4 h post derivatization compared to the initial injection (p less than 0.05); however, the acetyl derivative was stable for 24 hours and had a CV of less than 10% at a cutoff of 300 ng/mL.

Nuclear magnetic resonance studies of flexible opiate conformations at monoclonal antibody binding sites. Quantitative interproton distances obtained from comparing theoretical and experimental transferred nuclear Overhauser enhancement: correlation with antibody sequence.

A previous publication described the use of qualitative intramolecular 1H-transferred nuclear Overhauser effect measurements to determine the conformations of flexible ligands at monoclonal anti-opiate antibody binding sites. This paper concentrates on the quantitative interpretation of experiments of this type using the ligand nalorphine (N-allyl morphine) and a single anti-opiate monoclonal antibody. I compare the experimental unidimensional driven nuclear Overhauser effect buildup curves to theoretical curves derived with a knowledge of the fixed interproton distances in the ligand. The discussion covers the potential accuracies of derived distances and concentrates on two problem areas associated with determining structures from this type of experiment. The most serious one is the case where, because of particular multiproton spatial distributions, spin diffusion is so rapid that it cannot be determined experimentally and where numerical fits of theoretical calculations are misleading. The results show that, while intraligand spin diffusion complicates the interpretation for some proton pairs, with many others accuracies within about 0.3 A for interproton distances from 2 to 4 A are attainable. The results confirm the earlier report that the conformation of nalorphine in this antibody binding site differs from the major one present in solution or in the crystal. An important aspect of the work is that theoretical prediction of nuclear Overhauser effect time-dependence is an important practical tool for recognizing cases where interpretation of experiments will be difficult. Initial data on protein-to-ligand transferred nuclear Overhauser effect are presented, which show that at least one aromatic amino acid residue is closely involved in the binding of the ligand. The companion paper presents the primary sequences of the variable regions of the antibodies being used in our studies. In this paper, these and associated immunochemical studies are correlated with the nuclear magnetic resonance results. The combination of data presented in the two papers provides a basis for future work on protein-ligand interproton distances in the range 1 to 5 A using both transferred nuclear Overhauser effect (for rapidly exchanging ligands) and isotope-edited, indirectly detected nuclear Overhauser effect (for tightly bound ligands).

[Comparative morphine determination in parts of the brain using combined gas chromatography/mass spectrometry. A possibility for assessing survival time]. / Vergleichende Morphinbestimmung an Gehirnteilen mittels kombinierter GC/MS. Eine Möglichkeit zur Eingrenzung der Uberlebenszeit.

Comparison of the morphine concentrations in the medulla oblongata and the cerebellum provides information on the interval between morphine administration and death. If this period is short, the ratio of CMed:CCereb is below 1. The ratio is above 1 if a number of hours have passed at least. The balance of the morphine concentrations is achieved within a few hours. Results have been achieved by gas chromatography/mass spectrometry analysis in about 100 autopsy cases.

Liquid chromatography with pre-column dansyl derivatisation and fluorimetric detection applied to the assay of morphine in biological samples.

A simple method employing pre-column dansylation and liquid chromatography is proposed for a very sensitive and specific assay of morphine in biological samples. Nalorphine is used as an internal standard. The detection limit is 0.2 picomol of injected morphine. In the assay of human sera spiked with 150 nmol/l, the intra- and inter-assay coefficients of variation were 3.7% (n = 10) and 4.5% (n = 10), respectively. No interferences were observed from more than 70 opiate and non-opiate drugs. Urine, plasma and total blood were assayed, using different extraction methods, with negligible interference from coextractives.

Determination of opiates in biological samples by glass capillary gas chromatography with electron-capture detection.

A gas chromatographic method for the simultaneous determination of morphine, 6-acetylmorphine and codeine in human plasma or blood has been developed. The samples are buffered to pH 9 and extracted on silica columns, cleaned by extraction and finally acylated with pentafluoropropionic anhydride. The derivatives formed are separated on a glass capillary column with falling glass needle injection and electron-capture detection. The choice of the extraction conditions and the preparation of suitable capillary columns are discussed.

Separation of standard opiates and their analysis in pharmaceutical and illicit preparations by paired-ion reverse-phase high-pressure liquid chromatography.

The separation of standard opiates in a mixture and their analysis in clandestine and pharmaceutical preparations were accomplished by PIC on a micro-Bondapak C18 column. The identification of the opiates was based on two parameters: retention times and the ratios of absorbance peaks recorded at 254 and 280 nm. No prior clean-up procedure of samples was required for analysis by this method. Baseline separation of drug components in clandestine and in pharmaceutical preparations made this method suitable for their quantitation.

Radioimmunoassay of morphine and morphine-like substances in biological fluids and human tissues.

A radioimmunoassay is described for the determination of morphine and morphine-like substances in plasma, serum, biological fluids and tissue homogenates using an antiserum to morphine-6-hemisuccinyl-BSA and 125I-morphine as the labelled tracer. In the B/F separation with ammonium sulphate, calcium sulphate was added to make the precipitate more compact. Some parameters related to the use of this method in direct assay on plasma, serum, other biological fluids and tissue homogenates were evaluated.