François-Ludovic Sauvage

A Fully Automated Turbulent-flow Liquid Chromatography-tandem Mass Spectrometry Technique for Monitoring Antidepressants in Human Serum

Antidepressants belong to a variety of chemical and pharmacologic classes. Most require therapeutic drug monitoring, at least in certain circumstances, such as unexplained inefficacy or suspected toxicity. Several types of chromatographic methods have generally been used. This paper presents a fully automated, sensitive, and specific method for the therapeutic drug monitoring of 13 antidepressants of all classes (amoxapine, amitriptyline, citalopram, clomipramine, dothiepin, doxepin, fluoxetine, imipramine, maprotiline, mianserin, paroxetine, sertraline, trimipramine) and some of their respective active metabolites (nortriptyline, monodesmethylcitalopram, desmethylclomipramine, desipramine, norfluoxetine, desmethylmianserin, N-desmethylsertraline), based on the innovative turbulent-flow liquid chromatography (TFC) technology, coupled to tandem-mass spectrometry (MS/MS). The antidepressants were divided in two groups depending on their chromatographic properties, so that two injections would be necessary to screen all compounds (which is infrequent for therapeutic drug monitoring). Calibration curves ranged from 10 to 500 ng/mL. No significant memory effect was observed after the injection of a blank serum sample spiked at 500 ng/mL. The intra-assay and inter-assay precision CVs ranged from 0.4% to 12% and from 1% to 16%, respectively. The method was further validated by blindly analyzing Heathcontrol-Therapeutic Drugs Scheme samples (Cardiff Bioanalytical Services Ltd.) containing several antidepressants.

Pitfalls and Prevention Strategies for Liquid Chromatography-Tandem Mass Spectrometry in the Selected Reaction– Monitoring Mode for Drug Analysis

BACKGROUND We observed cases of false-positive results with the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Different LC-MS/MS techniques that use the selected reaction-monitoring mode, routinely employed for the analysis and quantification of drugs and toxic compounds in biological matrices, were involved in the false-positive and potentially false-positive results obtained. We sought to analyze the causes of and solutions to this problem. METHODS We used a previously reported LC-MS/MS general unknown screening method, as well as manual spectral investigation in 1 case, to perform verification and identification of interfering compounds. RESULTS We observed that false-positive results involved: a metabolite of zolpidem that might have been mistaken for lysergic acid diethylamide, benzoylecgonine mistaken for atropine, and clomipramine and 3 phenothiazines that share several common ion transitions. CONCLUSIONS To prevent problems such as those we experienced, we recommend the use of stable-isotope internal standards when possible, relative retention times, 2 transitions or more per compound when possible, and acceptable relative abundance ratios between transitions, with an experience-based tolerance of +/-15% for transitions with a relative abundance >10% and with an extension to +/-25% for transitions <10% when the concentration is at the limit of quantification. A powerful general unknown screening procedure can help to confirm suspected interferences. Our results indicate that the specificity of screening procedures is questionable for LC-MS/MS analyses performed in the selected reaction-monitoring mode and involving a large number of compounds with only 1 transition per compound.

Contribution of the different UDP-glucuronosyltransferase (UGT) isoforms to buprenorphine and norbuprenorphine metabolism and relationship with the main UGT polymorphisms in a bank of human liver microsomes.

The goal of this study was to evaluate the specific contribution of individual UDP-glucuronosyltransferase (UGT) isoforms in the metabolism of buprenorphine (BUP) and norbuprenorphine (Nor-BUP), as well as the impact of their genetic variations. The glucuronidation of BUP and Nor-BUP was examined using human liver microsomes (HLMs) and heterologously expressed UGTs. The individual contribution of UGT isoforms was estimated using enzyme kinetic experiments combined with the relative activity factor (RAF). Phenotype-genotype relationships were investigated in a bank of 52 HLMs. Among the six hepatic UGT isoforms tested, UGT1A1, UGT1A3, and UGT2B7 metabolized BUP and Nor-BUP. Using the RAF approach, we found that UGT1A1 and UGT2B7 accounted for approximately 10 and 41% of BUP glucuronidation, respectively. Nor-BUP glucuronidation involved predominantly UGT1A3 (approximately 63%) and UGT1A1 (34%), whereas UGT2B7 had only a minor role. The UGT1A1 promoter (TA)6/7TAA mutation (UGT1A1*28) resulted in a 28% decrease of BUP glucuronidation Vmax in pooled HLMs but was not statistically associated with glucuronidation rate in 52 individual HLMs. The presence of the UGT2B7 promoter (G–842A) mutation resulted in higher BUP glucuronidation Vmax in pooled HLMs (+80% on average) and in a significant higher glucuronidation rate in noncarriers (but not in carriers) of the UGT1A1*28 allele (P = 0.0352). This study represents a functional basis for further clinical pharmacogenetic studies.

Metabolism of Sirolimus in the Presence or Absence of Cyclosporine by Genotyped Human Liver Microsomes and Recombinant Cytochromes P450 3A4 and 3A5

Sirolimus is an immunosuppressive drug currently used alone or in combination with cyclosporine. Both drugs undergo extensive metabolism by the CYP 3A enzymes. This study aimed at comparing the activity of recombinant CYP (rCYP) 3A4 and 3A5 toward sirolimus, investigating the effect of cyclosporine on the metabolic rate of these two cytochromes P450 (P450s), as well as the impact of the CYP 3A5*3 polymorphism on that of human liver microsomes (HLMs). Two distinct approaches were used; i.e., the measurement of (1) hydroxy-sirolimus and desmethyl-sirolimus production, and (2) sirolimus depletion by the in vitro half-life method. rCYP 3A5 exhibited a lower intrinsic clearance (CLint) for both hydroxylation (0.11 versus 0.24 μl/pmol P450/min) and depletion of sirolimus (0.64 versus 2.36 μl/pmol P450/min) than rCYP 3A4. Similar CLint values for hydroxylation, demethylation, and depletion were found when comparing a pool of HLMs carrying at least one CYP 3A5*1 (active) allele with a pool of HLMs not expressing CYP 3A5. This was further confirmed for sirolimus depletion using individual microsome preparations (p = 0.42). A deeper inhibitory effect of cyclosporine on the CLint of sirolimus depletion was found for rCYP 3A4 than for rCYP 3A5 (i.e., –44% versus –8% at 0.62 μM, 750 μg/l cyclosporine), and sirolimus metabolism was slightly less inhibited for HLMs expressing CYP 3A5 than not (–38% versus –56%). In the absence of cyclosporine, the CYP 3A5*3 polymorphism may not influence significantly sirolimus metabolism at the hepatic level. However, strong CYP 3A4 inhibition by cyclosporine could unveil the influence of this polymorphism.

Involvement of UDP-Glucuronosyltransferases UGT1A9 and UGT2B7 in Ethanol Glucuronidation, and Interactions with Common Drugs of Abuse

Ethyl glucuronide (EtG) determination is increasingly used in clinical and forensic toxicology to document ethanol consumption. The enzymes involved in EtG production, as well as potential interactions with common drugs of abuse, have not been extensively studied. Activities of human liver (HLM), kidney (HKM), and intestinal (HIM) microsomes, as well as of 12 major human recombinant UDP-glucuronosyltransferases (UGTs), toward ethanol (50 and 500 mM) were evaluated in vitro using liquid chromatography-tandem mass spectrometry. Enzyme kinetic parameters were determined for pooled microsomes and recombinant UGTs with significant activity. Individual contributions of UGTs were estimated using the relative activity factor approach, proposed for scaling activities obtained with cDNA-expressed enzymes to HLM. Interaction of morphine, codeine, lorazepam, oxazepam, nicotine, cotinine, cannabinol, and cannabidiol (5, 10, 15 mg/l) with ethanol (1.15, 4.6, 11.5 g/l; i.e., 25, 100, 250 mM) glucuronidation was assessed using pooled HLM. Ethanol glucuronidation intrinsic clearance (Clint) was 4 and 12.7 times higher for HLM than for HKM and HIM, respectively. All recombinant UGTs, except UGT1A1, 1A6, and 1A10, produced EtG in detectable amounts. UGT1A9 and 2B7 were the most active enzymes, each accounting for 17 and 33% of HLM Clint, respectively. Only cannabinol and cannabidiol significantly affected ethanol glucuronidation. Cannabinol increased ethanol glucuronidation in a concentration-dependent manner, whereas cannabidiol significantly inhibited EtG formation in a noncompetitive manner (IC50 = 1.17 mg/l; inhibition constant (Ki) = 3.1 mg/l). UGT1A9 and 2B7 are the main enzymes involved in ethanol glucuronidation. In addition, our results suggest that cannabinol and cannabidiol could significantly alter ethanol glucuronidation.

Identification of Acepromazine in Hair: An Illustration of the Difficulties Encountered in Investigating Drug-facilitated Crimes

Abstract:  After a drug‐facilitated sexual assault (DFSA), a woman was found in a drowsy state at home. She remembered having drunk an unknown beverage by the accused. Blood samples (collected 8 hours after the DFSA), two glasses, and a teaspoon seized by the police were analyzed. Acepromazine, a phenothiazine tranquilizer used in human and veterinary medicine, was detected in the residue of one of the glasses. In spite of acepromazine absence in the victim’s blood, the possible use of acepromazine in the DFSA was reported to the police. Two weeks later, a suspect admitted having orally administered acepromazine to the victim. Using a liquid chromatography‐tandem mass spectrometry method, this compound was subsequently detected (31 pg/mg) in a sample of the victim’s hair collected a month and a half after the DFSA. A potential short elimination half‐life in humans and/or the well‐known in vitro degradation of acepromazine could explain the negative blood result. DFSA toxicological investigations are challenging and can be complicated when a rather unusual substance is concerned. In particular, special care should be taken when interpreting the results, taking into account elimination and/or instability data, when available.

Feasibility of Ribavirin Therapeutic Drug Monitoring in Hepatitis C

Ribavirin, a nucleoside analog, is administered in combination with interferon to patients with chronic hepatitis C. To evaluate the feasibility of ribavirin therapeutic drug monitoring, we investigated the influence of blood collection and preanalytical conditions on ribavirin concentrations and compared the results obtained from interlaboratory blind tests by 3 laboratories using different analytical techniques. On 3 occasions, blank serum samples spiked with ribavirin and pooled serum samples from patients on ribavirin were sent to the 3 laboratories. Two analytical techniques were based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) and 1 on high-performance liquid chromatography with UV detection (HPLC-UV), with protein precipitation or solid-phase extraction, all validated according to international guidelines. Inter- and intra-batch mean relative errors ranged from −7.4% to +10.3% and from −10.3% to +7.4%, respectively. Relative standard deviations were <13.5% and <10.6%, respectively. Linearity, assessed blindly, between 125 and 4550 ng/mL was excellent (r > 0.991) for all 3 methods. The 2 LC-MS/MS techniques were slightly less precise and accurate than HPLC-UV, perhaps because the internal standard used was not a ribavirin isotope. Accurate and precise LC-MS/MS and HPLC-UV methods developed in 3 different laboratories provided excellent and consistent results to blind tests for ribavirin determination in spiked serum samples and pools of serum samples from patients with chronic hepatitis C virus infection.

Stability of Ribavirin Concentrations Depending on the Type of Blood Collection Tube and Preanalytical Conditions

Ribavirin pharmacokinetic and exposure effect trials based on either plasma or serum concentrations have yielded diverging results. This study aimed to compare ribavirin concentrations in serum and plasma and to investigate the influence of blood collection and preanalytical conditions on ribavirin concentration stability. Blood samples from patients with hepatitis virus C and receiving ribavirin were collected in plain (dry) tubes, in tubes containing ethylenediaminetetra-acetic acid or lithium-heparinate, in Type II Serum Separating Tubes with clot activator, or Type II lithium heparinate Plasma Separating Tubes. Different time and temperature conditions were tested before and after blood centrifugation. Ribavirin was determined using liquid chromatography-dual mass spectroscopy. Multiple-way analysis of variance was used for statistical analyses. Ribavirin concentrations showed a higher interlaboratory variability in serum than in plasma. Results were fairly stable over 2 hours in whole blood collected in dry or ethylenediaminetetra-acetic acid tubes and very stable up to 24 hours in serum or plasma kept in gel-containing tubes after immediate centrifugation. When Plasma Separating II gel tubes were kept at +4°C or at ambient temperature for up to 24 hours before centrifugation, ribavirin concentrations decreased by 1% to 8% and 12% to 18%, respectively. These results suggest that blood samples should be collected in gel-containing tubes and centrifuged immediately, after which the tubes can be kept at ambient temperature for the next 24 hours. In case of clinical constraints, Plasma Separating II gel tubes can be kept at +4°C for a maximum of 2 hours before centrifugation with limited impact on the measured concentrations.

LC-MS/MS Screen for Xenobiotics and Metabolites

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is now considered as a perfect complement to HPLC-DAD (diode array detection) and gas chromatography (GC)-MS for the general unknown screening of drugs and toxic compounds.Here we describe a procedure applied routinely in our laboratory for clinical and forensic applications using the QTRAP™ technology.

Untargeted Screening of Urinary Peptides Using Offline Nano-Liquid Chromatography: MALDI-TOF/TOF Mass Spectrometry

In renal transplantation, the discovery of early urine biomarkers of graft lesions would be useful in helping physicians to improve patient care and minimize the use of invasive techniques such as biopsies. Over the last years, high-resolution mass spectrometry has been used extensively for the search of biomarkers in various biological fluids. Here we describe a procedure based on reverse-phase nano-HPLC, offline plate spotting, and MALDI-TOF and TOF/TOF applied in our laboratory for the search of natural peptides in urine samples from renal transplant patients.