Sensitivity Enhancement for Direct Injection Capillary Electrophoresis to Determine Morphine in Human Serum via In-capillary Derivatization

Journal of Chromatographic Science, 2019, Vol. 57, No. 2, 177–185 doi: 10.1093/chromsci/bmy092 Advance Access Publication Date: 1 October 2018 Article Article Sensitivity Enhancement for Direct Injection Capillary Electrophoresis to Determine Morphine in Human Serum via In-capillary Derivatization Samy Emara1,*, Walaa Zarad1, Maha Kamal2, Ahmed Ali3, and Yasmine Aboulella3 1 Pharmaceutical Chemistry Department, Faculty of Pharmacy, Misr International University, Km 28 Ismailia Road, Cairo 44971, Egypt, 2Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Modern Sciences and Arts University, 26 July Mehwar Road intersection with Wahat Road, 6 October City 12573, Egypt, and 3Laboratory for Single Cell Mass Spectrometry, RIKEN Quantitative Biology Center, 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan * Author to whom correspondence should be addressed. Email: Received 29 January 2018; Revised 11 August 2018; Editorial Decision 21 August 2018 Abstract Rapid and simple micellar electrokinetic chromatography (MEKC) with in-capillary derivatization and fluorescence detection has been developed to determine morphine in human serum. The sample was introduced into a background electrolyte (BGE) containing potassium ferricyanide, whereas morphine was oxidized into highly fluorescent product, pseudomorphine. Different parameters for derivatization and subsequent separation were systematically investigated for the analysis of morphine in serum. Efficient performance of the developed MEKC system was carried out in a single run using BGE made up of 70 mM sodium tetraborate decahydrate (pH 10.5), 0.30 mM potassium ferrricyanide, 80 mM sodium dodecyl sulfate, and applied voltage of 9 kV. The combination of MEKC with in-capillary derivatization of morphine was successfully achieved with a high degree of sensitivity. The validation of the method showed good linearity between areas of morphine and the corresponding concentrations over the range of 5-5000 ng/mL. Excellent accuracy and precision were obtained at all concentration levels. The mean recoveries of morphine were ranging from 83.86 to 94.45%. The validated MEKC method successfully permitted determination of morphine in clinical samples after a single oral dose of controlled release morphine sulfate tablets. Introduction Morphine is a relatively strong opioid that is routinely used to manage acute and chronic pain (1). Despite its effectiveness in pain relief, it may cause serious side effects which necessitate close monitoring of its levels in biological fluids routinely (2). Several analytical techniques, such as gas chromatography (GC) (3–6) and highperformance liquid chromatography (HPLC) (7–15), have been employed to detect and quantify morphine in biological fluids. However, these methods have drawbacks such as lengthy analysis time and tedious dispensing steps for sample preparation, which makes them inconvenient and increase the chances of error. Moreover, extensive derivatization before analysis is essential in most cases of GC. In an attempt to eliminate these problems, immunological assays such as radioimmunoassay (16) and enzyme immunoassay (17) were the preferred methods for therapeutic drug monitoring, because they were rapid, specific, sensitive and could analyze biological fluids directly without prior sample pretreatment procedures. Unfortunately, immunoassay methods are only limited © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: 177 178 three groups: zone-passing (28), at-inlet (29) and through the electrophoretic solutions containing the reagents (30). Intact morphine possesses low native fluorescence; however, it can be oxidized into highly fluorescence pseudomorphine by reaction with ferricyanide in an alkaline medium (13, 31–33). Accordingly, derivatization of morphine on-line into a highly fluorescent product before performing analysis in biological fluids is needed. For this type of derivatization, in-capillary mode was a better choice for MEKC and fluorescence detection of morphine in serum. With a more efficient therapeutic application of various drugs and the need to examine their concentrations in body fluids for diagnostic and research purposes, there has evolved the need for reliable and automated analytical procedures. Therefore, it was important to investigate the potential of MEKC for the determination of morphine in human serum and to establish suitable conditions for automated derivatization and separation in a single run without any sample pretreatment step. The applicability of the developed method to practical serum samples was also emphasized in the present work. Experimental Instrumentation and general procedure Separation was performed in a fused-silica capillary covered with a polyamide-coating layer (Polymicro Technologies, Phoenix, AZ, USA). The total length of the capillary was 90 cm, and the length from the inlet end to detection point was 80 cm with 75 μm i.d. and 360 μm o.d. The detector was a FP-920 fluorescence detector (Jasco), equipped with the flow cell unit for HPLC replaced by the capillary cell unit for CE (Jasco, Tokyo, Japan). The electropherograms were recorded by monitoring the fluorescence intensity at 450 nm (excited at 340 nm). Both ends of the capillary were separately dipped in the anodic and cathodic solutions, having the same composition as the BGE, and the surface of these electrode solutions were adjusted to the same level. The system is equipped with A model HCZE-30 PNO 25-LDS stabilized high voltage power supply to apply voltage up to 30 kV (Matsusada Precision Devices, Japan). The high-voltage end of the capillary was enclosed in an acrylic glass enclosure as a safety precaution. The CE system was operated using normal polarity (the cathode was located on the detector side). Injection was done in a hydrodynamic fashion by dipping the injection end of the capillary in the sample reservoir for 12 s while it rested 15 cm above the cathodic end reservoir. After the sample was introduced, the capillary was placed back into the BGE vial, and a potential of 9 kV was applied between both ends of the capillary, to move morphine for derivatization and to transport the resulting fluorescent product to the detector. Electropherograms were processed and recorded on a chromatopack integrator C-R6A (Shimadzu, Kyoto, Japan). The CE instrument was operated at ambient temperature (22°C ± 1°C). The CE system used a BGE consisting of 70 mM sodium tetraborate decahydrate (pH 10.5), 0.30 mM potassium ferricyanide and 80 mM SDS. The electrophoretic mobility of morphine and the first serum peak was calculated according to the formula: μ = [(L t /tm ) − (L t /teo)]/(V /Ld ), where μ is the electrophoretic mobility of the analyte, tm the migration time measured directly from the electropherogram, teo the to specific drugs and can be impaired by specific and non-specific interferences. Capillary electroph (...truncated)