Kristian Linnet

Simultaneous screening and quantification of 52 common pharmaceuticals and drugs of abuse in hair using UPLC–TOF-MS

An UPLC-TOF-MS method for simultaneous screening and quantification of 52 drugs in hair was developed and validated. The selected drugs represent the most common classes of pharmaceuticals and drugs of abuse such as amphetamines, analgesics, antidepressants, antipsychotics, benzodiazepines, cocaine, ketamine and opioids. Hair samples were extracted with methanol:acetonitrile:ammonium formate (2 mM, 8% acetonitrile, pH 5.3) overnight at 37 degrees C. The target drugs were separated and quantified using a Waters ACQUITY UPLC coupled to a Waters Micromass LCT Premier XE Time-of-Flight mass spectrometer. Total chromatographic run time was 17min. The data were treated with the MassLynx software ChromaLynx XS and QuanLynx for automated identification and quantification, respectively. The limits of detection ranged from 0.01 to 0.10 ng/mg using a 10-mg hair sample and the limit of quantification was 0.05 ng/mg for 87% of the analytes. A good linear behaviour was achieved for most of the analytes in the range from LOQ to 10 or 25 ng/mg except for the amphetamines. The method showed an acceptable precision and trueness, since the obtained CV and BIAS values were <or=25% for 81% of the analytes. The extraction recoveries for 92% of the analytes ranged between 84 and 106% and the extraction recoveries for all analytes were better than 60%. The method was applied to 15 autopsy hair samples from forensic investigations showing a wide abuse pattern of many pharmaceuticals and drugs of abuse within a period of less than three months. The present study demonstrated that the combination of accurate mass and retention time can provide good selectivity, which demonstrates that the TOF instrument is adequate for both screening and quantification purposes. Furthermore, it was shown that screening with the ChromaLynx XS software is less sensitive and selective for some analytes than the QuanLynx software, especially in low concentrations.

A review on the impact of P-glycoprotein on the penetration of drugs into the brain. Focus on psychotropic drugs

In recent years there has been increasing focus on the role of the drug transporter P-glycoprotein (P-gp) with regard to drug penetration into the brain. Studies using mice devoid of functional P-gp have revealed that P-gp at the blood-brain barrier (BBB) can exert a profound effect on the ability of some drugs to enter the brain, e.g. cardiovascular drugs (digoxin, quinidine), opioids (morphine, loperamide, methadone), HIV protease inhibitors, the new generation of antihistamines, and some antidepressants and antipsychotics. Among the latter group, risperidone is strongly influenced having about 10 times higher cerebral concentration in P-gp knock-out mice than in control mice. Taking into account that polytherapy is commonplace in psychiatry, theoretically there is a risk of drug-drug interactions with regard to P-gp at the BBB. Here we review the evidence for a role of P-gp with regard to psychoactive drugs from in vitro studies and experiments in knock-out mice devoid of functional P-gp. Moreover, the evidence for significant drug-drug interactions involving psychotropic drugs in rodents is considered. Clinical observations suggesting a role for P-gp in relation to drug-drug interactions at the BBB are sparse, and a definite conclusion awaits further studies. Also, the possible clinical relevance of P-gp genetic polymorphisms is questionable, and more investigations are needed on this subject.

Simultaneous Screening and Quantification of 29 Drugs of Abuse in Oral Fluid by Solid-Phase Extraction and Ultraperformance LC-MS/MS

BACKGROUND The European DRUID (Driving under the Influence of Drugs, Alcohol And Medicines) project calls for analysis of oral fluid (OF) samples, collected randomly and anonymously at the roadside from drivers in Denmark throughout 2008-2009. To analyze these samples we developed an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for detection of 29 drugs and illicit compounds in OF. The drugs detected were opioids, amphetamines, cocaine, benzodiazepines, and Delta-9-tetrahydrocannabinol. METHOD Solid-phase extraction was performed with a Gilson ASPEC XL4 system equipped with Bond Elut Certify sample cartridges. OF samples (200 mg) diluted with 5 mL of ammonium acetate/methanol (vol/vol 90:10) buffer were applied to the columns and eluted with 3 mL of acetonitrile with aqueous ammonium hydroxide. Target drugs were quantified by use of a Waters ACQUITY UPLC system coupled to a Waters Quattro Premier XE triple quadrupole (positive electrospray ionization mode, multiple reaction monitoring mode). RESULTS Extraction recoveries were 36%-114% for all analytes, including Delta-9-tetrahydrocannabinol and benzoylecgonine. The lower limit of quantification was 0.5 mug/kg for all analytes. Total imprecision (CV) was 5.9%-19.4%. With the use of deuterated internal standards for most compounds, the performance of the method was not influenced by matrix effects. A preliminary account of OF samples collected at the roadside showed the presence of amphetamine, cocaine, codeine, Delta-9-tetrahydrocannabinol, tramadol, and zopiclone. CONCLUSIONS The UPLC-MS/MS method makes it possible to detect all 29 analytes in 1 chromatographic run (15 min), including Delta-9-tetrahydrocannabinol and benzoylecgonine, which previously have been difficult to incorporate into multicomponent methods.

Serum concentrations and side effects in psychiatric patients during risperidone therapy.

Steady state serum concentrations of risperidone and 9-hydroxyrisperidone (9-OH-risperidone), the active moiety, were measured in 42 patients. The concentration-to-dose ratios (C/D) varied by a factor of 20, from 1.8 to 36.8 (nmol/l)/(mg/24 hours), and 90% of the active moiety was constituted of 9-OH-risperidone. No correlation between the serum concentration of the active moiety and the side effects evaluated by the UKU Side Effect Scale was found. The absence of CYP2D6 (poor metabolizers) or the coadministration of drugs other than benzodiazepines increased the ratio between parent compound and metabolite but did not significantly influence the C/D of the total active moiety. A therapeutic range for serum risperidone has not been established, but 6 mg/day is considered the optimum dose for most patients. The authors found that in 90% of 22 patients administered 6 mg/day risperidone, the serum levels were within 50 to 150 nmol/l.

Olanzapine serum concentrations in psychiatric patients given standard doses: the influence of comedication.

We recorded steady state serum concentrations of olanzapine in 56 psychiatric patients under routine conditions. Twenty-two patients (39%) underwent monotherapy; the rest received additional psychotropic drugs. Doses were given once daily in the evening, and serum olanzapine levels were measured 12 hours later. For the whole group, the concentration-to-dose ratio (C/D) varied 26-fold, with a median value of 4.8 (nmol/L)/(mg/24 hours), but 80% of the patients had C/D values within the range 2 to 10 (nmol/L)/(mg/24 hours). All but three patients received standard doses (5-20 mg/24 hours), of whom 80% had serum concentrations of olanzapine within the range 22 to 146 nmol/L. Patients comedicated with potential inhibitors of CYP2D6 and other drugs displayed a median C/D approximately 40% higher than the group undergoing monotherapy. Patients comedicated with carbamazepine had a median C/D 36% lower than that of the monotherapy group. Therefore, the serum concentration range (12-hour values) of 25 to 150 nmol/L can be expected for patients receiving a standard daily dose.

Contributions of five human cytochrome P450 isoforms to the N-demethylation of clozapine in vitro at low and high concentrations.

The authors assessed the in vitro contribution of cytochrome P450 (CYP) isoforms 1A2, 3A4, 2C9, 2C19, and 2D6 to the N‐demethylation of clozapine mediated by human liver microsomal preparations (HLM). In contrast to previous studies, the authors focused on a relatively low hepatic concentration level, 5 pM, to assess the conditions at a therapeutically relevant hepatic concentration level of clozapine. The optimal concentrations of specific inhibitors were ini‐tially established using cDNA‐expressed CYP isoforms. The mean contributions of CYPs 1A2, 2C19, 3A4, 2C9, and 2D6 amounted to 30%, 24%, 22%, 12%, and 6%, respectively, with regard to the total HLM‐mediated N‐demethylation.

Fluvoxamine-Clozapine drug interaction: inhibition in vitro of five cytochrome P450 isoforms involved in clozapine metabolism.

Administration of fluvoxamine to patients receiving clozapine therapy may increase the steady-state serum concentrations of clozapine by a factor of 5 to 10. The authors undertook in vitro studies to disclose the mechanism behind this clinically important interaction. In a human liver microsome preparation, fluvoxamine showed a concentration-dependent inhibition of clozapine N-demethylation. Fluvoxamine was much less effective as an inhibitor of clozapine N-oxidation. Fluvoxamine also inhibited in a concentration-dependent manner the activity of all five cytochrome P450 (CYP) isoforms previously determined to be capable of catalyzing the demethylation of clozapine. Fluvoxamine inhibited CYP1A2 and 2C19 with the highest affinities (Ki values of 0.041 and 0.087 microM, respectively). The Ki values for CYP2C9 and 2D6 were 2.2 and 4.9 microM, respectively, whereas the Ki for CY3A4 was 24 microM. Assuming a hepatic tissue concentration of fluvoxamine in the range of 4 to 7 microM under therapeutic conditions, a clinically significant inhibition of all but CYP3A4 is expected in relation to clozapine N-demethylation. No significant effect of fluvoxamine on clozapine N-oxidation is to be expected under therapeutic conditions. Because of the large interindividual variability of the quantity of the various CYP isoforms in liver tissue, it is not possible to predict the fluvoxamine-induced increase in the plasma concentration of clozapine of an individual patient.

Quantifying the Accuracy of a Diagnostic Test or Marker

BACKGROUND In recent years, increasing focus has been directed to the methodology for evaluating (new) tests or biomarkers. A key step in the evaluation of a diagnostic test is the investigation into its accuracy. CONTENT We reviewed the literature on how to assess the accuracy of diagnostic tests. Accuracy refers to the amount of agreement between the results of the test under evaluation (index test) and the results of a reference standard or test. The generally recommended approach is to use a prospective cohort design in patients who are suspected of having the disease of interest, in which each individual undergoes the index and same reference standard tests. This approach presents several challenges, including the problems that can arise with the verification of the index test results by the preferred reference standard test, the choice of cutoff value in case of a continuous index test result, and the determination of how to translate accuracy results to recommendations for clinical use. This first in a series of 4 reports presents an overview of the designs of single-test accuracy studies and the concepts of specificity, sensitivity, posterior probabilities (i.e., predictive values) for the presence of target disease, ROC curves, and likelihood ratios, all illustrated with empirical data from a study on the diagnosis of suspected deep venous thrombosis. Limitations of the concept of the diagnostic accuracy for a single test are also highlighted. CONCLUSIONS The prospective cohort design in patients suspected of having the disease of interest is the optimal approach to estimate the accuracy of a diagnostic test. However, the accuracy of a diagnostic index test is not constant but varies across different clinical contexts, disease spectrums, and even patient subgroups.

Citalopram and desmethylcitalopram concentrations in breast milk and in serum of mother and infant

A case is presented with measurements of the selective serotonin reuptake inhibitor (SSRI)-antidepressant citalopram in serum of mother and breast-fed infant and in breast milk. We found a milk-to-serum concentration ratio of approximately 3 for both citalopram and the main metabolite desmethylcitalopram. Peak milk concentrations of citalopram occurred 3-9 h after drug intake by the mother. The infant received approximately 5% of the mothers dose, adjusted for weight. Accordingly, the serum level in the infant of approximately 7 nM corresponded to approximately 1/15 of the trough serum concentration of the mother (104 nM). No signs of drug effects in the infant were observed.

Screening for illicit and medicinal drugs in whole blood using fully automated SPE and ultra‐high‐performance liquid chromatography with TOF‐MS with data‐independent acquisition

A broad forensic screening method for 256 analytes in whole blood based on a fully automated SPE robotic extraction and ultra-high-performance liquid chromatography (UHPLC) with TOF-MS with data-independent acquisition has been developed. The limit of identification was evaluated for all 256 compounds and 95 of these compounds were validated with regard to matrix effects, extraction recovery, and process efficiency. The limit of identification ranged from 0.001 to 0.1 mg/kg, and the process efficiency exceeded 50% for 73 of the 95 analytes. As an example of application, 1335 forensic traffic cases were analyzed with the presented screening method. Of these, 992 cases (74%) were positive for one or more traffic-relevant drugs above the Danish legal limits. Commonly abused drugs such as amphetamine, cocaine, and frequent types of benzodiazepines were the major findings. Nineteen less frequently encountered drugs were detected e.g. buprenorphine, butylone, cathine, fentanyl, lysergic acid diethylamide, m-chlorophenylpiperazine, 3,4-methylenedioxypyrovalerone, mephedrone, 4-methylamphetamine, p-fluoroamphetamine, and p-methoxy-N-methylamphetamine. In conclusion, using UHPLC-TOF-MS screening with data-independent acquisition resulted in the detection of common drugs of abuse as well as new designer drugs and more rarely occurring drugs. Thus, TOF-MS screening of blood samples constitutes a practical way for screening traffic cases, with the exception of δ-9-tetrahydrocannabinol, which should be handled in a separate method.