Determination of methylphenidate in plasma and saliva by liquid chromatography/tandem mass spectrometry

Abstract

Methylphenidate (MPH) is a phenethylamine derivative used in the treatment of attention-deficit hyperactivity disorder (ADHD). In adults, clinical monitoring of MPH therapy is usually performed by measuring plasma MPH concentrations. In children blood sampling is however undesirable. Saliva may be an alternative matrix for monitoring MPH concentrations with the advantage that it can be obtained non-invasively. Therefore, we developed an analytical method for the quantification of MPH in both plasma and saliva. We present the validation of a liquid chromatography–tandem mass spectrometric method using a hydrophilic interaction liquid chromatography column (HILIC). In 100 μL sample, proteins were precipitated with 750 μL acetonitrile/methanol 84/16 (v/v) containing d9-methylphenidate as the internal standard. Standard curves were prepared over the MPH concentration range of 0.5–100.0 μg/L. The total analysis time was 45 s. Accuracy and within- and between-run imprecision were in the range of 98–108% and less than 7.0%, respectively. Matrix effects were greater for plasma than saliva with 46% and 8% ionization suppression. The matrix effects were adequately compensated by the use of deuterated MPH as internal standard. MPH significantly degraded in plasma and saliva at room temperature and 5 °C. Samples were stable at −20 °C for at least 4 weeks. The method was successfully applied for the determination of MPH concentrations in plasma and saliva samples from an adult healthy volunteer. Using protein precipitation and hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry, this method allows fast, accurate and precise quantification of MPH in both plasma and saliva.

Introduction

Methylphenidate (MPH) is a psychostimulant widely used in the treatment of attention-deficit hyperactivity disorder (ADHD). ADHD is a neurobehavioral problem mostly encountered in school-aged children at a prevalence of 5–10% of the general population [1], [2]. MPH is a piperidine-derived molecule that contains two chiral centers and exists as four stereoisomers (Fig. 1). The pharmacological activity resides entirely with the dl-threo-methylphenidate racemic (50:50) mixture [3].

The major metabolic pathway of MPH is the hydrolysis of the methyl ester linkage by esterases to form ritalinic acid [4], [5]. Minor metabolic pathways for both these compounds include parahydroxylation of the aromatic ring, oxidation to 6-oxo-dervatives and glucuronide formation [6], [7]. Ritalinic acid and the other metabolites are pharmacologically inactive [8], [9], [10].

There is a clinical need to perform therapeutic drug monitoring (TDM) in patients who are undergoing MPH therapy. MPH exhibits wide inter-individual variability in both pharmacokinetics and clinical response [11], [12]. TDM can be applied when the patient remains unresponsive to therapy, exhibits unexpected adverse events or to check adherence. In adults, clinical monitoring of MPH therapy is usually performed by measuring plasma MPH concentrations. In children, monitoring of drug levels should be performed with minimal discomfort for the patient. Monitoring of MPH concentrations in saliva may therefore be an interesting non-invasive alternative to blood sampling as demonstrated earlier by Marchei and coworkers [13].

Several methods have been developed for quantification of MPH in plasma, urine and hair, using high-performance liquid chromatography (HPLC) with ultraviolet detection [6], [14], capillary electrophoresis-mass spectrometry [15], gas chromatography–mass spectrometry [8], [9], [16] and liquid chromatography–tandem mass spectrometry [7], [13], [17], [18]. The determination of MPH concentrations by standard reversed-phase (RP) chromatography coupled to MS/MS detection is particularly challenging since retention times may be short due to the high hydrophilicity of the compound. This may produce a significant loss in sensitivity due to the co-elution with matrix interference and the high percentage of water at the chromatographic elution time. Recent research however has shown that for hydrophilic compounds the sensitivity, precision and accuracy of a quantitative analytical chromatographic method may be improved by using hydrophilic interaction liquid chromatography (HILIC) [19]. In addition, the use of HILIC has advantages in sample preparation when measuring polar compounds. Because of the high organic modifier content, usually acetonitrile, used during chromatography, proteins can be precipitated using organic solvents without the loss of chromatographic integrity, as is often the case when used with polar compounds in combination with RP chromatography. A high organic modifier concentration is also ideal for compound ionization by electrospray ionization mass spectrometry (ESI-MS), resulting in higher sensitivity.

The aim of the present study was to develop a method to determine MPH concentrations in human plasma and saliva for potential use in therapeutic drug monitoring. We present the development and validation of an analytical method using HILIC chromatography coupled to tandem mass spectrometry. The stability of MPH in plasma and saliva was investigated at different temperatures. The applicability of the method was demonstrated with plasma and saliva data from one healthy adult volunteer obtained before and after intake of 10 mg immediate release (IR) MPH and 18 mg MPH – osmotic controlled-release oral delivery system (OROS) – on different occasions.