Determination of Common Antipsychotics in Quantisal-Collected Oral Fluid by UHPLC-MS/MS: Method Validation and Applicability for Therapeutic Drug Monitoring.

BACKGROUND: Oral fluid (OF) is an interesting alternative for conventional blood testing in therapeutic drug monitoring. OF can be used for screening but its value for quantification has to be established. METHODS: To evaluate the value of OF for quantification of 11 commonly used antipsychotics (APs) and 5 metabolites, an ultra-high performance liquid chromatography-tandem mass spectrometric method was validated. OF was obtained from psychiatric patients using a Quantisal collection device. OF to serum concentration ratios were determined, taking into account the exact volume of collected OF. RESULTS: Linearity was evaluated at 7 or 8 calibration levels. Accuracy criteria were fulfilled, except for pipamperone (PIP) at quality control (QC) low. The intraday precision ranged 0.88%-14.73% and interday precision ranged 1.92%-16.17%. The mean recovery from the collection pad was 37.1% at QC low and 40.3% at QC high for 1 mL of collected OF; for 0.5 mL collected OF mean recovery was 35.0% at QC low and 37.3% at QC high. When 0.1 mL OF was collected, recovery data were unreliable. Mean absolute matrix effect was 101.1% (82.0%-120.0%). OF patient samples (n = 89) containing 269 APs and metabolites were acquired and the mean volume of collected OF was 0.562 mL (0.057-1.232 mL). The OF to serum ratios were above 1 for all APs (1.54-28.50), except for aripiprazole (0.21) and zuclopenthixol (0.66). A broad range of calculated ratios for all APs was obtained. CONCLUSIONS: This validated ultra-high performance liquid chromatography-tandem mass spectrometric method can be used to reliably quantify APs in OF, even when recovery is low. Because the correlation between OF and serum concentrations was low and in addition results were highly variable, it can only be concluded that OF is a potentially interesting matrix, particularly for screening for noncompliance.

Ethyl glucuronide concentrations in hair: a controlled alcohol-dosing study in healthy volunteers.

Ethyl glucuronide (EtG) is a minor phase II metabolite of alcohol that accumulates in hair. It has been established as a sensitive marker to assess the retrospective consumption of alcohol over recent months using a cut-off of ≥7 pg/mg hair to assess repeated alcohol consumption. The primary aim was to assess whether amounts of alcohol consumed correlated with EtG concentrations in hair. Additionally, we investigated whether the current applied cut-off value of 7 pg/mg hair was adequate to assess the regular consumption of low-to-moderate amounts of alcohol. A prospective controlled alcohol-dosing study in 30 healthy individuals matched on age and gender. Individuals were instructed to drink no alcohol (N = 10), 100 g alcohol per week (N = 10) or 150 g alcohol per week (N = 10) for 12 consecutive weeks, before and after which hair was collected. Throughout the study, compliance to daily alcohol consumption was assessed by analyzing urine EtG three times weekly. Participants in the non-drinking group had median EtG concentrations of 0.5 pg/mg hair (interquartile range (IQR) 1.7 pg/mg; range < 0.21-4.5 pg/mg). Participants consuming 100 and 150 g alcohol per week showed median EtG concentrations of 5.6 pg/mg hair (IQR 4.7 pg/mg; range 2.0-9.8 pg/mg) and 11.3 pg/mg hair (IQR 5.0 pg/mg; range 7.7-38.9 pg/mg), respectively. Hair EtG concentrations between the three study groups differed significantly from one another (p < 0.001). Hair EtG concentrations can be used to differentiate between repeated (low-to-moderate) amounts of alcohol consumed over a long time period. For the assessment of repeated alcohol use, we propose that the current cut-off of 7 pg/mg could be re-evaluated.

Therapeutic drug monitoring of common antipsychotics.

The aim of this review is to provide information for interpreting outcome results from monitoring of antipsychotics in biological samples. A brief overview of the working mechanisms, pharmacological effects, drug interactions, and analytical methods of classical and atypical antipsychotics is given. Nineteen antipsychotics were selected based on their importance in the worldwide market as follows: amisulpride, aripiprazole, asenapine, bromperidol, clozapine, flupenthixol, haloperidol, iloperidone, lurasidone, olanzapine, paliperidone, perphenazine, pimozide, pipamperone, quetiapine, risperidone, sertindole, sulpiride, and zuclopenthixol. A straightforward relationship between administered dose, plasma or serum concentration, clinical outcome, or adverse effects is often lacking. Nowadays, focus lies on therapeutic drug monitoring and individualized therapy to find adequate treatment, to explain treatment failure or nonresponse, and to check patient compliance. However, extensive research in this field is still mandatory.

Qualitative and semi-quantitative screening of selected psychoactive drugs in blood: Usefulness of liquid chromatography - triple quadrupole and quadrupole time-of-flight mass spectrometry in routine toxicological analyses.

Routine toxicological analysis requires broad screening for a large number of therapeutically prescribed and other compounds, and/or their metabolites. This article specifically focuses on three classes of psychoactive substances: antidepressants (ADs), antipsychotics (APs) and benzodiazepines and Z-drugs (BZDs). Two screening methods were compared for their ease-of-use in a routine setting, based upon the analysis of 105 medico-legal case samples. Analytes of interest were extracted using liquid-liquid extraction and separated using liquid chromatography with a total run time of 12 min per sample. A first detection method used targeted triple quadrupole mass spectrometry, operated in triggered multiple reaction monitoring mode (tMRM). False negative results were noted for 15% of the total number of detected analytes only, the majority of which were either present at sub- to low therapeutic levels or were metabolites of other analytes in the samples. The occurrence of false positive results was rare. A second screening method used quadrupole time-of-flight mass spectrometry (QTOF) for untargeted data acquisition. Data analysis was facilitated by the creation of an in-house, subset mass spectral database. As was seen for the tMRM screening, false negative results were observed in less than 20% of the total number of detected analytes, either for compounds at low concentrations or of which metabolites could be identified in the samples. More false positive results were observed due to an observed bias for prothipendyl. Determination of the exact concentration in a sample may only be required depending on the specific case circumstances. For this purpose, semi-quantification using each of the screening methods was investigated. Excellent results were observed using the tMRM method in combination with a small number of labelled internal standards (n = 12). Semi-quantification using the QTOF screening method was more laborious, but limited results on selected compounds indicated equally good results. Overall, the developed semi-quantitative screening methods performed well and - following further validation on case samples - could be implemented for most compounds in routine toxicological analysis without the need for highly trained or specialised personnel.

Multi-analyte LC-MS/MS quantification of 38 antipsychotics and metabolites in plasma: Method validation & application to routine analyses.

The past decades have seen a rise in the prescription of antipsychotic drugs in the European population, despite the risk of extra-pyramidal, metabolic and cardiac side effects. A multi-analyte liquid chromatography - triple quadrupole mass spectrometry method was developed for the quantification of 38 antipsychotic drugs in plasma. Samples were extracted by a straightforward liquid-liquid extraction with methyl-tertiary-butyl-ether and the compounds of interest were chromatographically separated within 6 min. Calibration curves covered the recommended therapeutic range for all compounds, in addition to sub- and supratherapeutic concentrations for most. The method was successfully validated according to the European Medicines Agency guidelines on bioanalytical method validation. Analysis of medico-legal samples confirmed the relatively common use of the second generation antipsychotics quetiapine and olanzapine, as well as the continued presence of the first generation antipsychotic haloperidol.

Quantification of 54 Benzodiazepines and Z-Drugs, Including 20 Designer Ones, in Plasma.

Benzodiazepines are widely used in the treatment of sleep and anxiety disorders, as well as epileptic seizures and alcohol withdrawal because of their broad therapeutic index and low cost. Due to their central nervous system depressant effects they are also often implicated in traffic accidents and drug-related intoxications. With an increasing number of designer benzodiazepines used in a recreational setting, there is a need for analytical methods to be able to quantify both the prescribed and designer benzodiazepines. A liquid chromatography-triple quadrupole mass spectrometry method was developed for the quantification of 34 prescribed and 20 designer benzodiazepines in plasma. Different sample preparation strategies, including protein precipitation, liquid-liquid extraction, solid-phase extraction and mini-QuEChERS, were tested. The best recoveries for all compounds of interest were obtained with a liquid-liquid extraction using methyl-tertiary-butyl-ether and 500 µL plasma. The method was fully validated according to the European Medicines Agency guidelines for all compounds, except pivoxazepam, which is included for qualitative purposes only. In-sample stability issues were observed for cloxazolam, both at ambient temperature and during long-term storage at -20°C. Due to the large number of compounds included, the simple and time-efficient sample preparation and the relatively inexpensive instrumentation used, the presented method can be readily implemented in both therapeutic drug monitoring and forensic analyses.

Optimization of a liquid chromatographic separation for the simultaneous determination of four anthracyclines and their respective 13-S-dihydro metabolites.

The optimization of a liquid chromatographic separation of four anthracyclines (doxorubicin, epirubicin, daunorubicin and idarubicin) and their respective 13-S-dihydro metabolites (doxorubicinol, epirubicinol, daunorubicinol and idarubicinol) is described. Inclusion of epidaunorubicin in the assay allows internal standardization. Potential chromatographic interference of aglycones (doxorubicinone, daunorubicinone, idarubicinone, doxorubicinolone, daunorubicinolone and idarubicinolone) was investigated and tackled. The analyses were performed on a C18 RP column and gradient elutions were performed with a mixture of 0.1% formic acid in water and 0.1% formic acid in ACN. The analytes were measured by fluorescence detection with an excitation wavelength of 480 nm and emission wavelength of 555 nm, respectively. The separation will potentially allow a broad field of applications, ranging from therapeutic drug monitoring and kinetic studies in cancer patients to monitoring hospital personnel and effluents.

Validation of a simple, fast liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of 40 antidepressant drugs or their metabolites in plasma.

INTRODUCTION: Antidepressant (AD) use has increased significantly over the last decades. Therapeutic drug monitoring is recommended for compliance, toxicity and treatment efficiency. ADs also show a high prevalence in forensic cases. Few methods have been developed that combine a fast, easy sample clean-up with a quantification based on liquid chromatography-triple quadrupole mass spectrometry (LC-QQQ). METHODOLOGY: A liquid-liquid extraction (LLE) was performed using 200 µL of plasma. The evaporated and reconstituted upper fraction was injected on a LC-QQQ system monitoring 3 transitions per compound. The method was fully validated according to international guidelines. RESULTS & DISCUSSION: The chromatographic run time was under 12 min. The LLE was successful in removing interferences with minimal sensitivity loss. Calibration curves ranged from sub-therapeutic to toxic concentrations. Quality control samples showed high accuracy (81%-119%) and precision (≤14%) within and between batches. Stability was tested at ambient temperature and -20 °C. The method was successfully applied to external quality control and case samples. CONCLUSION: The presented method successfully quantifies 40 compounds of interest. Because of a simple sample clean-up, a relatively short chromatographic run and a wide calibration range this method can be implemented in therapeutic drug monitoring, forensic research and related fields.

Traces of phosgene in chloroform: consequences for extraction of anthracyclines.

Chloroform is commonly used to extract anthracyclines from various biological matrices. However, their determination can be seriously compromised by phosgene traces present as a result of failing stabilization of chloroform. Out of the three varieties in which chloroform exists (not stabilized, stabilized with an alcohol and stabilized with a hydrocarbon) only the ethanol stabilized type minimizes chances on creating artifacts. Chromatographic separation after extraction of four anthracyclines (doxorubicin, epirubicin, daunorubicin and idarubicin) and two metabolites (13-S-dihydrodoxorubicin and 13-S-dihydroepirubicin) with chloroform under various conditions indicate that the appropriate choice of stabilizer in this extraction solvent is highly relevant.

Development of a solid phase extraction for 13 'new' generation antidepressants and their active metabolites for gas chromatographic-mass spectrometric analysis.

A solid phase extraction procedure (SPE) for 13 'new' antidepressants (venlafaxine, fluoxetine, viloxazine, fluvoxamine, mianserin, mirtazapine, melitracen, reboxetine, citalopram, maprotiline, sertraline, paroxetine and trazodone) together with eight of their metabolites (O-desmethylvenlafaxine, norfluoxetine, desmethylmianserine, desmethylmirtazapine, desmethylcitalopram, didesmethylcitalopram, desmethylsertraline and m-chlorophenylpiperazine) from plasma is optimized using HPLC-DAD as monitoring system. Special attention has been paid to the choice of washing and eluting solvent, resulting in a highly concentrated, clean and moisture free extract, also suitable for GC-MS. A total number of 10 sorbents (apolar, polymeric, ion-exchange and mixed mode) was evaluated. Based on recovery, reproducibility and absence of interfering substances the strong cation exchanger gave the best results. Recoveries were determined at low and high therapeutic and toxic levels and ranged between 70 and 109% for all compounds, except for trazodone (39%).