Hollow fiber liquid-phase microextraction combined with ultra-high performance liquid chromatography–tandem mass spectrometry for the simultaneous determination of naloxone, buprenorphine and norbuprenorphine in human plasma

https://doi.org/10.1016/j.jchromb.2014.01.029Get rights and content

Highlights

  • Buprenorphine, norbuprenorphine and naloxone were quantified in plasma by UPLC–MS/MS.

  • This is the first extraction of these analytes from human plasma by HF–LPME (PVDF).

  • All the extraction conditions of HF–LPME were optimized carefully.

  • This method was compared with SPE method to prove the reliability.

  • This method is contributed to pharmacokinetic studies of BP, NBP and NLX.

Abstract

A hollow fiber liquid phase microextraction (HF–LPME) combined with ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed for the extraction and determination of naloxone (NLX), buprenorphine (BP) and its major metabolite norbuprenorphine (NBP) in human plasma. The optimum extraction conditions of HF–LPME were: the porous of polyvinylidene fluoride (PVDF) hollow fiber was full of component solvent (1-octanol/chloroform/toluene, 2/4/4), the pH of donor phase was 8.7, the extraction time was 30 min and stirring speed was 1000 revolutions per minute (rpm). The UHPLC–MS/MS method was performed with Waters ACQUITY UPLCTM BEH C18, 50 mm × 2.1 mm, 1.7 μm, using methanol–0.2%formic acid as mobile phase with a gradient elution at a flow rate of 0.25 mL/min. The target compounds were detected under a tandem quadrupole mass spectrometer in positive electrospray ionization (ESI) mode, then analyzed in multiple reaction monitoring (MRM) mode and the isotope internal standard method was used for quantification. The results showed that linearities were in the range of 0.1–25 ng/mL (R > 0.996). The limits of detection (LOD) of BP/NBP/NLX were 0.05/0.05/0.025 ng/mL and the limits of quantitation (LOQ) of BP/NBP/NLX were 0.1/0.1/0.05 ng/mL, respectively. The spiked recoveries were in the range of 92.1–106.0% with relative standard deviation (RSD) values were less than 15%. This method was simple, inexpensive, sensitive and has been successfully used to quantify plasma samples from patients included in a clinical pharmacogenetic study.

Introduction

Buprenorphine (BP) is a derivative of the morphine alkaloid thebaine and it has been available worldwide as a parenteral and sublingual analgesic since the 1970s. The analgesic effect of BP is stronger than morphine 25 to 50 times [1], but it is able to produce dependence and addiction [2]. BP is commercialized as a sublingual tablet as a pure substance or associated with naloxone (NLX) to prevent diversion to intravenous use [3]. The clinical use of BP for the treatment of chronic pain is limited, however BP abuse is becoming increasingly common worldwide[4], [5]. Some drug addicts took BP as a substitute for heroin. BP and NLX sublingual (4:1) dose formulation may decrease parenteral BP abuse. Norbuprenorphine (NBP) is one of primary metabolites which is formed by demethylating buprenorphine by CYP3A4 in the liver and intestinal wall. Due to a low therapeutic concentration of the drug in body fluids, the quantitation of BP in biological materials requires highly sensitive analytical techniques, gas chromatography–mass spectrometry (GC–MS) [6], liquid chromatography–mass spectrometry (LC–MS) [7], [8], [9], liquid chromatography–tandem mass spectrometry (LC–MS/MS) [10], [11], capillary electrophoresis (CE) [12] had been reported for the determination of BP in the biological samples.

Sample preparation for biological samples analysis is necessary because of low analyte concentration, complex sample matrices and limited available sample volumes. Most procedures use liquid–liquid extraction (LLE) [13] and solid-phase extraction (SPE) [11] prior to LC–MS, GC–MS or CE. LLE offers high reproducibility and high sample capacity, while LLE uses large amounts of samples, toxic and expensive high-purity organic solvents, and its extraction process is time-consuming, tedious and easily produce emulsions. Consumption of organic solvents is relatively low in SPE, however, SPE is expensive and its still requires lengthy process (i.e. conditioning, washing, eluting, and drying). These drawbacks might be overcome by using hollow fiber liquid-phase microextraction (HF–LPME).

Pedersen-Bjergaard and Rasmussen introduced hollow fiber liquid-phase microextraction [14]. HF–LPME combines extraction, concentration and sample clean-up in one step. Hollow fiber membrane can preserve the acceptor phase from the interference of sample solution. Some large molecules (proteins and compounds) can not be transferred from the donor phase to the acceptor phase. Two phases and three phases are the two main types of HF–LPME. In three phases type hollow fiber liquid–liquid–liquid microextraction, the pores of the hollow fiber are full of the organic phase and the lumen of the fiber is filled with the acceptor phase. The target analytes in their non-ionized form can be extracted from the sample into the organic phase. The analytes are subsequently extracted into the acceptor phase with a pH that is adjusted to ionize the analytes. The basic principles of this technique have been described in previous reviews [15], [16], [17], [18]. One of advantages of HF–LPME is its tolerance to a wide pH range. Moreover, the hollow fiber for preparation of each sample is cheap, so the cost could be reduced and sample carry-over can be avoided by making them affordable to dispose of after a single use [19]. For low levels target determination or complex biological samples analysis, there has been a highly interest in HF–LPME [18]. Additional advantage of HF–LPME method is compatible with most of analytical instruments such as GC, high performance liquid chromatography (HPLC) and CE [20], [21]. Up to now, porous hydrophobic membranes in general are most commonly used for membrane extraction purposes, such as polypropylene, polytetrafluoroethylene (PTFE) and polyvinylidene difluoride [22]. Usually, PP fiber is most commonly used in previous works in HF–LPME field [23]. Recently, PVDF fiber has been widely used in HF–PLME, due to its good mechanical strength, thermal and chemical stability, higher porosity, better solvent compatibility and fast extraction efficiency [24], [25].

Usually, SPE was used for the sample preparation of human plasm containing buprennorphine and its metabolite[26], [27], [28]. In order to reduce the cost of the preparation of samples, the HF–LPME sample preparation method was developed and compared with the SPE method to verify the feasibility [29].

In this study, a HF–LPME (PVDF hollow fiber) combined with UHPLC–MS/MS was applied for the extraction and simultaneous determination of naloxone, buprenorphine and norbuprenorphine in human plasma. All the parameters of HF–LPME such as the nature of the immobilized organic phase, pH of donor phase, extraction time and stirring rate have been optimized. Finally, the developed and validated method was applied to the determination of the target compounds in plasma samples. In addition, in order to evaluate the applicability of this method in pharmacokinetic studies, the plasma samples collected from some healthy volunteers submitted to single dose treatment with buprenorphine and naloxone sublingual (4:1) [30]. The results compared with SPE method show that HF–LPME can be used in the plasm sample preparation.

Section snippets

Chemicals and solutions

BP, BP-D4, NBP, NBP-D3, NLX-D5 in methanol solutions (each 100 μg/mL) were purchased from Cerilliant (Austin, TX, USA). NLX was purchased from Sigma (St. Louis, MO, USA). Methanol and formic acid (both HPLC grade) were obtained from Fisher (USA). Chloroform, 1-octanol, toluene, n-hexane and acetone were purchased from Beijing Chemical Plant. Water was deionized to a resistivity of more than 18.2 MΩ with a Milli-Q ultrapure water system (Millipore Corp., Woburn, MA, USA).

Materials

Polypvinylidene fluoride

Optimization experimental conditions for HF–LPME extraction

The extraction efficiency of the device may be affected by organic solvents, donor phase pH, stirring speed, extraction time and salt addition effect. The recovery was selected as analytical response during optimization process. Measurements were taken in three replicates.

Conclusions

It is the first time that a HF–LPME (PVDF hollow fiber) technique followed by UHPLC–MS/MS was used for preconcentration and determination of BP, NBP and NLX in human plasma samples. All the extraction conditions of HF–LPME were optimized carefully. Wide linear range of this method can be satisfactorily applied for BP, NBP and NLX in therapeutic drug monitoring. By comparing with SPE method, HF–LPME is a simple, inexpensive method for extraction and preconcentration of BP, NBP and NLX from

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