Development and validation of a liquid chromatography–mass spectrometry assay for hair analysis of methylphenidate

Abstract

Methylphenidate (MPH) is a phenethylamine derivative used in the treatment of childhood attention-deficit hyperactivity disorder. MPH is biotransformed in the body by the hydrolysis of the methyl ester linkage to its metabolite, ritalinic acid. Whereas both compounds are usually measured in plasma and urine, preliminary observations show that only the parent compound is present in hair from treated individuals.

Since in children hair samples can be easily collected without the need for specials skills and exposing a patient to discomfort, hair testing of MPH should be an alternative to check compliance in a wider time-window than if using blood.

A procedure based on liquid chromatography–mass spectrometry (LC–MS) has been developed for the determination of MPH in hair of treated children. After addition of 3,4-methylenedioxypropylamphetamine as internal standard, hair samples were overnight digested with 0.1 M HCl at 37 °C. Then, after pH adjustment to 6 using 1N NaOH, and 0.1 M phosphate buffer, the analyte was extracted with Bond–Elut Certify columns. Chromatographic separation was achieved at ambient temperature using a reverse phase column and a mobile phase of 80% 10 mM ammonium acetate–20% acetonitrile with a 20 min gradient program. The mass spectrometer was operated in positive electrospray ionization and selected ion monitoring acquisition mode.

The method was validated in the range 0.15–50 ng MPH/mg hair, using 20 mg hair per assay. At three concentrations spanning the linear dynamic range of the assay, mean recoveries ranged between 73.2 and 77.1%. First results show MPH hair concentration varying from 0.15 to 4.17 ng/mg hair, with decreasing drug concentration in distal hair segments, even in children treated with the same MPH dose during the period corresponding to different segments. This fact could be either attributed to sebum or sweat shunt with the most proximal hair segment or drug degradation by cosmetic treatments in more distal segments.

Introduction

Attention-deficit hyperactivity disorder (ADHD) is the most common neurobehavioural disorder of childhood affecting school-aged children, with a prevalence generally estimated to be 5–10% of general population [1], [2], [3]. Although multimodal treatment approaches are advocated, pharmacotherapy with psychostimulants remains a cornerstone of treatment for ADHD [2], [4], [5]. Of the available medications, methylphenidate (MPH), a phenethylamine derivative, is the most extensively studied and widely prescribed [6], [7]. MPH is reported to be absorbed quickly and completely from the gut after oral administration [8] and it is rapidly hydrolyzed in the methyl ester linkage to its metabolite, ritalinic acid [6], [8]. Minor metabolic pathways for both these compounds include parahydroxylation of the aromatic ring, oxidation to 6-oxo-derivates and glucuronide formation [9]. Both MPH and ritalinic acid are usually measured in plasma and urine [6], [8], [9], but therapeutic drug monitoring for this drug is essentially lacking and early studies reported that assay of MPH blood levels did not appear to be clinically useful in the management of hyperactive children [10].

It is known that there is marked individual variability in the dose–response relationship for MPH, and therefore dosage must be titrated for optimal effect and avoidance of toxicity in each child. It is unclear whether this variability is predominantly pharmacokinetic or pharmacodynamic [11].

Monitoring exposure to therapeutic drugs in pediatric population is more difficult to perform than in adults because the need for non-invasive or less invasive, yet highly sensitive, analytical methods to assess active or passive drugs intake. This fact stimulated, in the last decades, the application of hair analysis in pediatrics [12]. Since in children hair samples can be easily collected without the need for specials skills and exposing a patient to discomfort, hair testing of MPH should be an alternative to check compliance in a wider time-window than if using blood.

Several analytical methods have been reported for the determination of MPH in plasma and urine. Most of these procedures are based on ultraviolet detectors [9], [13], capillary electrophoresis–mass spectrometry [14], gas chromatography–mass spectrometry [6], [8], [15] and liquid chromatography–tandem mass spectrometry [6]. Differently, the determination of MPH and its metabolite in hair has been never investigated. Within the framework of a study concerning the use of alternative biological matrices in therapeutic drug monitoring in pediatrics, we aimed to look for the presence of MPH and ritalinic acid in hair from treated children and eventual application of hair testing for compliance monitoring. In accordance with the fact that acidic substances are seldom incorporated in hair matrix, preliminary observations showed that ritalinic acid is absent in hair from treated children. For this reason, we focused on detection of parent drug solely in keratin matrix. In the present paper, we report the development and validation of a liquid chromatography (LC) electrospray ionization (ESI)–mass spectrometry (MS) method for determination of MPH in hair from treated children.