Behavioural Parameters of Circadian Rhythm Are Not Correlated with Dim Light Melatonin Onset: An Observational Study on Healthy Volunteers.

Dim light melatonin onset (DLMO) is considered the most reliable marker of the circadian rhythm phase in humans. DLMO may moderately correlate with sleep onset and sleep offset time. There are no sufficient data about the correlations between DLMO and clinical scales assessing sleep quality and daytime symptoms of poor night sleep. The aim of the study was to determine the association between DLMO and basic sleep parameters from actigraphy and sleep diaries, as well as the association between DLMO and the following insomnia clinical scales: the Athens Insomnia Scale (AIS), Insomnia Severity Index (ISI), Epworth Sleepiness Scale (ESS), and chronotype questionnaires: Morningness-Eveningness Questionnaire (MEQ) and Composite Scale of Morningness (CSM). Participants of the study were healthy volunteers. Sleep parameters were measured by sleep diaries and actigraphy, and the following clinical scales: the AIS, ISI, and ESS, and chronotype questionnaires: MEQ and CSM. DLMO was calculated based on plasma melatonin concentration. The blood samples were collected hourly at five time points between 20:00 and 00:00 during the session in dim red light (<50 lux). Melatonin concertation was determined by LC-MS/MS. Twenty-one volunteers participated in the study. DLMO was calculated in 12 participants. There was a significant correlation between DLMO and ISI (r = 0.60, p = 0.038) and ESS (r = 0.61, p = 0.034). The correlation coefficient between the DLMO and the AIS was also high, however insignificant (r = 0.57, p = 0.054). There were no significant correlations between DLMO and chronotype scales MEQ and CSM. DLMO did not correlate with sleep onset and sleep offset; however, DLMO correlated with the Sleep Fragmentation Index (SFI) (r = 0.67, p = 0.017). DLMO is associated with poorer sleep maintenance, a stronger feeling of insomnia, and sleepiness during the day. Simultaneously, chronotype pattern and circadian rhythm parameters do not correlate with DLMO. Biological circadian rhythm does not reflect the real-life sleep-wake rhythm, indicating that the lifestyle is more often disconnected from the biological clock.

Assessment of dim light melatonin onset based on plasma and saliva samples.

Melatonin (MELA) is a nocturnal hormone involved in the regulation of the circadian rhythm. MELA can be detected in plasma and saliva, and its salivary concentration strongly correlates with its plasma concentration. Dim light melatonin onset (DLMO) is considered to be the most accurate objective marker for assessing the circadian phase. The purpose of the study was to establish a method for the determination of MELA in plasma and saliva based on the liquid chromatography with tandem mass spectrometry (LC-MS/MS) and compare DLMO using both plasma and saliva matrices. The validation of the LC-MS/MS methods was performed in accordance with the European Medicines Agency (EMA) guideline. The study was conducted on a group of 21 volunteers, male and females, aged 26-54 years. Plasma and saliva were collected at five time points: between 20:00 and 00:00 hours. The MELA concentration was determined by the LC-MS/MS. The DLMO was considered as the point in time when MELA concentration exceeds 20 pg/mL in plasma and 7 pg/mL in saliva. The correlation coefficient between the plasma and salivary MELA concentration was r = 0.764 (p < .001). The ratio of the plasma/saliva MELA concentrations was 2.87. The mean time of the DLMO in the plasma was 21:30 ± 0:45 hours, and in the saliva was as follows: 21:34 ± 1:00 hours. The correlation between the DLMO, calculated based on the plasma and saliva MELA profiles, was r = 0.679 (p < .05). The determination of salivary MELA concentration using LC-MS/MS allows for the determination of the DLMO. Our method may be applied in clinical practice for the diagnosis and monitoring of circadian rhythm disorders.Abbreviations: CE: Collision Energy; CID: Collision-Induced Dissociation; DL: Desolvation Module; DLMO: Dim Light Melatonin Onset; EFSA: European Food Safety Authority; EMA: European Medicines Agency; ESI: electrospray ionization; HB: heat block; HPLC: high performance liquid chromatography; IS: internal standard; K3EDTA: ethylenediaminetetraacetic acid tripotassium salt; LC-MS/MS: liquid chromatography with tandem mass spectrometry; LLE: liquid-liquid extraction; LLOQ: lower limit of quantification; MELA: melatonin; MELA-D4: melatonin-d4; MRM: multiple reaction monitoring; Q1: quadrupole 1; Q3: quadrupole 3; RE: relative error; RIA: radioimmunoassay; RSD: relative standard deviation; SD: standard deviation; ULOQ: upper limit of quantification.

Hair as a matrix in monitoring drug epilepsy therapy.

Hair is considered an efficient tool to investigate drug-related histories; thus, the selection of the method of sample preparation is important to obtain a reliable result. The aim of this study was to compare two methods of hair preparation (cutting and pulverizing) to analyse levetiracetam concentration in hair. An additional aim was to evaluate the potential usefulness of the levetiracetam concentration measured as an index of a dosing schedule. Four groups of 12 rats were included in the experiment. Depending on the group, the rats received 10 mg/kg of levetiracetam intraperitoneally every 24, 48 and 72 hours for 30 days. The control group was not treated. At the end of the drug administration, the rats' hair was shaved, cut or pulverized and analysed by the LC/MS-MS method to determine the concentration of levetiracetam. A stronger correlation between the mean hair levetiracetam concentration in hair and the number of drug doses was found in pulverized hair than in cut hair. A smaller standard deviation between the results was obtained in the case of pulverized hair. The results indicate that pulverization gives a more reliable result of drug concentration in hair than cutting and that drug concentration in hair can reflect the scheme of levetiracetam administration.

Levetiracetam concentration in hair is associated with the time schedule of administration: Study on rats.

Measurement of hair drug content may be a reliable biomarker of the history of drug exposure, allowing to assess patient long-term compliance. Studies on correlations between antiepileptic drugs intake and their hair contents are limited. The aim of the study was to determine the association between the history of levetiracetam administration and its content in rat hair in controlled laboratory conditions. Additionally, the effects of levetiracetam on hair growth rate and body-weights were examined. Three groups of 12 rats each were exposed to different schedules of levetiracetam administration (10 mg/kg i.p.: every 24, 48 and 72 hours) for 30 days. The control group received daily saline injection. Levetiracetam concentrations in hair were assessed by validated LC-MS/MS method. The mean hair concentrations were as follows: 300 (±100), 170 (±60) and 130 (±80) ng/mg for rats receiving levetiracetam every 24, 48 and 72 hours, respectively. The levetiracetam accumulation in the hair was correlated with the total number of doses received (r = .699, P < .001). Levetiracetam did not affect the hair growth rate and rat body-weight. The concentration of levetiracetam measured in rat hair represents a reliable marker. It may reflect the adherence to levetiracetam treatment; however, further studies on human beings are needed.

Comparison of plasma, saliva, and hair lamotrigine concentrations.

BACKGROUND: In some clinical situations (pregnancy, aging, drug resistance, toxicity), measurements of lamotrigine plasma levels may be reliable. Limited studies indicate that saliva and hair could be alternative sources for monitoring lamotrigine therapy. The drug content in hair can also be used to assess the history of drug therapy and to ascertain long-term patient compliance. The aims of this study were to 1) determine the correlations among plasma, saliva, and hair lamotrigine concentrations, 2) evaluate saliva as an alternative matrix for monitoring drug levels and 3) evaluate hair as a source of information on adherence to antiepileptic treatment and on the correlation of hair concentrations with clinical outcomes in patients with epilepsy. METHODS: Plasma, saliva, and hair lamotrigine concentrations were measured by liquid chromatography-tandem mass spectrometry in positive ionization mode. The study group (n = 85) was recruited among the epileptic patients at the Institute of Psychiatry and Neurology, Warsaw, Poland. RESULTS: Plasma concentrations were not influenced by sex, age, or the concomitant use of other antiepileptic drugs. Lamotrigine saliva and plasma concentrations were strongly correlated (r = 0.82, p < 0.001). Lamotrigine hair concentrations were correlated with the plasma concentrations (r = 0.53, p < 0.001) and daily dose in mg/kg (r = 0.23, p = 0.024). The analysis revealed no significant correlation between lamotrigine hair levels and the number of seizures in the previous 3 months (r = -0.1, p > 0.05). CONCLUSIONS: The lamotrigine saliva concentration is strongly correlated with its plasma level, and saliva can be used as an alternative matrix to plasma for monitoring. Lamotrigine can also be successfully measured in hair, and the drug levels in hair tend to be correlated with the levels in plasma. However, lamotrigine levels in hair may not correspond to clinical outcomes (i.e., seizure episodes).