Journal of Chromatography B, 951–952 (2014) 157–163

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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 Wenjun Sun, Shuping Qu, Zhenxia Du ∗ Beijing University of Chemical Technology, Beijing 100029, China

a r t i c l e

i n f o

Article history: Received 20 October 2013 Received in revised form 12 January 2014 Accepted 20 January 2014 Available online 29 January 2014 Keywords: Buprenorphine Naloxone Norbuprenorphine HF–LPME UHPLC–MS/MS

a b s t r a c t 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 ␮m, 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. © 2014 Published by Elsevier B.V.

1. 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

∗ Corresponding author. Tel.: +86 1064433909. E-mail address: [email protected] (Z. Du). 1570-0232/$ – see front matter © 2014 Published by Elsevier B.V.

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) [7–9], 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).


W. Sun et al. / J. Chromatogr. B 951–952 (2014) 157–163

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 [15–18]. 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[26–28]. 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.

2. Experimental 2.1. Chemicals and solutions BP, BP-D4 , NBP, NBP-D3 , NLX-D5 in methanol solutions (each 100 ␮g/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 M with a Milli-Q ultrapure water system (Millipore Corp., Woburn, MA, USA).

2.2. Materials Polypvinylidene fluoride hollow fiber (1.15 mm external diameter, 0.80 mm inner diameter 0.16 ␮m pore size, 0.82 membrane porosity) was purchased from Institute of Biological and Chemical Engineering in Tianjin Polytechnic University. 2.3. Instrumentation Waters AcquityTM ultra-high performance liquid chromatography and Waters Micromass Quattro Premier XE triple quadrupole spectrometer (Waters, USA), which was equipped with an electrospray ion source using the multiple reaction monitoring (MRM) mode, were used in the present study. Instrument control and data acquisition were performed with MassLynx software V4.1 (Waters). Column used was Waters AcquityTM UPLC BEH C18 (50 mm × 2.1 mm, 1.7 ␮m) from Waters (USA). A mixture of methanol (solvent A) and 0.2% formic acid (solvent B) (5:95, v/v) were at a flow rate of 0.25 mL/min. An initial 5% solvent A was linearity increased to 95% in 5 min and then linearity decreased from 95% to 5% in 6 min; finally 5% solvent A was kept for reequilibration in 7.5 min. The injection volume was 10 ␮L in full loop mode. Column temperature was set at 30 ◦ C and autosampler temperature kept 10 ◦ C. The mass spectrometer was operated in MRM mode with positive electrospray ionization (ESI+ ) source. Compared with selected ion reaction (SIR) mode, MRM mode had more advantages in sensitivity and specificity for low concentrations of BP, NBP and NLX in plasma. In MRM mode, the most abundant ion was used for the quantitative ion (MRM1). The other ion was selected as confirmation of individual analytes (MRM2). Each compound’s cone voltage and collision energy were shown at Table 1. The nebulization gas was set at 600 L/h at a temperature of 350 ◦ C. The cone gas was set at 50 L/h. The capillary voltage was 3.5 kV and source temperature at 110 ◦ C. 2.4. Extraction procedure Hollow fiber was cleaned in acetone for 3 min in order to remove any contaminants. Before using, the hollow fiber was cut manually into 4 cm segments for each extraction and immersed in the mixed organic solvent (1-octanol/chloroform/toluene, 2/4/4) to fill the pores of the hollow fiber in 10 s. We found that the mixed organic solvent infiltrate the pores completely in less 10 s and the hollow fiber was almost transparent. Then, 20 ␮L of mixed organic solvent was injected to the hollow fiber lumen with a 100 ␮L HPLC syringe to avoid air bubble formation. After injection, the ends of the fiber were sheathed by a pair of mechanical crimping and sealed with a tweezer at the same time. BP, NBP and NLX were extracted under the following conditions: 1-octanol/chloroform/toluene (2/4/4) was used as the organic solvent; sample volume was 5 mL; stirring speed was 1000 rpm; extraction time was 30 min at 20 ◦ C. Experimental setup of HF–LPME was showed in Fig. 1. In this experiment, the samples were processed in parallel on two identical apparatus. After extraction, the fiber was taken out and washed outer wall of fiber by ultrapure water with a dropper. The fiber was put into a microcentrifuge tubes and add 500 ␮L (MeOH/H2 O, 1/1), rotated for 30 s. A 10 ␮L portion of the extractant was injected into the UHPLC–MS/MS system. 2.5. Preparation of plasma samples Blank human plasma was provided by healthy donors. The healthy human plasma used for spiking during method

W. Sun et al. / J. Chromatogr. B 951–952 (2014) 157–163


Table 1 Partial parameters of MRM detection. Compounds

MRM1 *


468.6 > 396.5 414.6 > 187.3* 328.6 > 310.5* 472.3 > 400.6* 417.7 > 187.3* 333.6 > 315.5*

CVa /CEb (V/eV)


CV/CE (V/eV)

70/40 55/40 30/19 65/40 55/40 30/15

468.6 > 101.2 414.6 > 101.2 328.2 > 253.3 472.3 > 101.2 417.7 > 101.3 333.6 > 212.4

70/42 55/40 30/24 65/42 55/40 30/35

Quantification ions. CV: Cone voltage. CE: Collision energy.

development was obtained from Beijing Anding Hospital. All the human plasma samples were stored at −20 ◦ C. Real plasma sample was obtained from 12 healthy volunteers. The sample solution (1.0 mL human plasma and 4.0 mL pure water) was vortexed for 30 s, sonicated for 2 min in glass tube before HF-LPME. 10 ␮L mixed internal standard (1.0 ng/mL) was added before the HF–LPME extraction.

however both are easily volatile, in order to prevent volatile loss of extract solvents and keep extraction solvents steadily retaining in the pores of the hollow fiber, 1-octanol was added into mixture solvents of chloroform and toluene, different ratios of 1octanol/chloroform/toluene were evaluated, the results showed that 1-octanol/chloroform/toluene (2/4/4) solvent with highest recoveries for three targets, so 1-octanol/chloroform/toluene (2/4/4) was set as optimum organic solvent by HF–LPME.

3. Results and discussion 3.1. 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. 3.1.1. Selection of organic solvents It is very crucial for HF–LPME extraction to select a suitable organic extraction solvent. A suitable organic solvents should be of such properties: low volatility to avoid solvent loss during the extraction process, appropriate viscosity to keep the solvent retain steadily in the pores of the hollow fiber, good affinity for the target compound to provide large enrichment factor and immiscibility with plasma. Based on the above considerations, different organic solvents were chosen to evaluate their influence on the extraction efficiency, including 1-octanol, chloroform, n-hexane, tributyl phosphate, toluene and their mixtures with different proportions. The results were shown in Fig. 2(a). N-hexane and tributyl phosphate have a poor extraction for all the compounds. Single 1octanol was not good for NLX extraction. Chloroform and toluene were able to get higher recoveries for NBP, NLX and BP respectively,

Fig. 1. Experimental setup of HF–LPME.

3.1.2. Optimization of donor phase pH The appropriate donor phase pH is also a very important parameter for extraction efficiency, it can change the partition coefficient of analyte between the sample solution and extraction solvent. In order to keep the analytes from being ionized in sample solution, we reduced the water solubility and increased extractability. A suitable amount of NaOH solution was added into the sample to adjust the pH before HF-LPME extraction. A series of different pH values were from 7.0 to 13.5. The results showed donor phase pH 8.5 was suitable for BP/NBP extraction and pH 9.0 for NLX in Fig. 2(b). But the recoveries of BP/NBP decreased sharply with donor phase pH 8.5 to 9.5. As the donor phase pH increases, transfer process through hollow fiber wall hole will be hindered by the plasma protein. Overall consideration, the highest extraction efficiency was obtained for the compounds at pH 8.6 which was chosen as the optimum value. 3.1.3. Optimization of extraction time and stirring speed In order to investigate the effect of extraction time on the extraction recovery, various extraction times in the range of 5–50 min was investigated in Fig. 2(c). Results indicated that the recoveries of BP/NBP/NLX were increased by increasing the extraction time up to 30 min and extended time would give rise to the possibility of organic solvent loss and lead to poor accuracy. Based on these observations, 30 min was selected as the optimum extraction time for the next experiments. Stirring of solution increases mass transfer in the donor phase and also reduces the time required to reach the thermodynamic equilibrium. Unfortunately, high stirring speed generates some problems such as acceleration of solvent evaporation and formation of air bubbles on the surface of the hollow fiber. To obtain optimal stirring speeds, different stirring speeds ranging from 0 to 1500 rpm were examined. Results in Fig. 2(d) showed that the stirring speed of 1000 rpm yielded the highest recovery (72%) and it was selected for subsequent experiments. 3.1.4. Effect of salt addition to the sample solution The effect of salt addition on the recoveries of BP/NBP/NLX extraction was evaluated by addition of NaCl from 0, 0.1 and 1.0 mg/mL in sample solution. Results in Fig. 2(e) showed that addition of NaCl had an adverse effect on recovery, so no salt was added into the sample solution in the subsequent experiments. It may be thought that in the presence of salt, interaction may take place between the analyte and salt. The interaction would tend to restrict the movement of analyte from donor phase to the membrane


W. Sun et al. / J. Chromatogr. B 951–952 (2014) 157–163

Fig. 2. The results of optimization experimental conditions for HF–LPME extraction. ((a) Organic solvents, (b) optimization of donor phase pH, (c) optimization of extraction time (min), (d) stirring speed (rpm), (e) effect of salt addition)

solvent or physical properties of the Nernst diffusion film changed by high concentration of salt. Because of salt, the rates of the diffusion of targets into organic solvent were reduced.

3.2. Method validation To evaluate the analytical performance of the HF–LPME– UHPLC–MS/MS method, specificity, limits of detection (LOD), linearity, matrix effect, recovery, repeatability and precision were investigated under optimal extraction conditions.

3.2.1. Specificity The specificity of the method was evaluated with respect to endogenous interferences by extracting and analyzing blank samples with the above-mentioned method. As can be seen from Fig. 3, no interference of endogenous compound was observed.

3.2.2. Matrix effect Matrix effect (ME) was used to describe the effect of matrix to the analyte ionization efficiency. The group A was made by a series of different concentrations and internal standard diluted with the mobile phase; the group B was made by a series of different concentrations and internal standard diluted with blank biological samples extracted by HF–LPME. Compared with the group A, response of the group B may reflect the matrix effect. ME (%) was calculated as follows: ME (%) = B/A × 100. No matrix effect is thought when ME (%) is equal to 100% ± 15%. Values over 115% or below 85% indicate ionization enhancement or suppression, respectively. ME (%) of BP, NBP and NLX were 72.18, 76.47 and 100.39, respectively. This experiment indicated ion suppression effect for BP, NBP and no matrix effect for NLX. In order to compensate for the whole method variability, including HF–LPME extraction and matrix effects caused by ion effects, this paper used stable isotope labeled of the objective compounds as internal standards.

W. Sun et al. / J. Chromatogr. B 951–952 (2014) 157–163


Fig. 3. The chromatography of blank plasma (A) and spiked plasma (B).

3.2.3. Linearity of calibration and limits of quantitation and detection Calibration was studied by analyzing blank plasma (1 mL) spiked with 10 ␮L of stock solutions with different mixing concentrations. The calibration standards were also spiked with 10 ␮L of ISs solution to prepare concentrations of 1.0 ng/mL of plasma for BPD4 , NBP-D3 and NLX-D5 , respectively. Blank plasma samples were extracted by HF–LPME and then analyzed by UPLC–MS/MS. Correlation coefficients for all compounds were greater than 0.9960.

LODs and LOQs were determined as the lowest concentration with a signal to noise ratio of at least 3:1 and 10:1 for the quantifier transition, respectively. The results were shown in Table 2. 3.2.4. Trueness, intra-assay, inter-assay variations and enrichment factors The quality control (QC) samples for intra-assay and interassay variations were prepared by spiking 1 mL blank plasma with 0.1 ng of each internal standard and the three different levels

Table 2 Standard curves of BP, NBP and NLX. Compound

Linear range (ng/mL)

Standard curve

Linear correlation coefficient (R)

LOD (ng/mL)

LOQ (ng/mL)


0.1–25 0.1–25 0.1–25

Y = 0.6269X − 0.001219 Y = 0.1347X − 0.0037 Y = 0.1939X + 0.0080

0.9973 0.9969 0.9979

0.050 0.050 0.025

0.100 0.100 0.050


W. Sun et al. / J. Chromatogr. B 951–952 (2014) 157–163

Table 3 The comparison between HF–LPME and SPE (n = 5) in the stability and sensitivity. Compound Cadded (ng/mL)


HF-LPME Cfound (ng/mL) RSD (%) Accuracy (%) SPE Cfound (ng/mL) RSD (%) Accuracy (%)

0.27 5.2 108 0.24 6.2 96





2.54 8.4 101.6 2.89 7.3 115.6



19.65 4.3 98.3 21.20 3.9 106


0.26 6.1 104 0.23 7.9 92


2.53 9.2 101.2 2.63 5.6 105.2


18.41 6.0 92.1 22.35 4.5 111.8

0.26 10.5 104 0.28 8.7 112

2.5 2.35 6.5 94 2.73 6.1 109.2

20 19.06 9.7 96 18.63 9.6 93.2

Table 4 The comparison between HF–LPME and SPE (each sample). Sensitivity (ng/mL)





0.05 0.05

0.05 0.25

0.025 0.025

Organic solvent volume (mL)

Time used (min)

Expenditure (RMB)


≤0.5 ≥5

30 60

5 25

In parallel In parallel

concentrations of analytes, followed by HF–LPME extraction and analyzed as described in Section 2.3. Each level of concentration was operated for six samples in parallel. The trueness of the three compounds varied from 92.0% to 110.0% with the relative standard deviation (RSD) lower than 15%. Precision should be within ±15% (LLOQ ± 20%). The enrichment factors for analytes were investigated in the optimum conditions. Enrichment factor was calculated by the ratio of the concentration (amount) of final analyte in the acceptor phase versus the concentration (amount) of final analyte in the donor phase. The enrichment factors of BP, NBP and NLX were 174, 179 and 213. 3.2.5. Recoveries Recoveries of the analytes were measured by spiking blank plasma samples (1 mL) with BP, NBP and NLX at three different concentrations: 0.25, 2.5 and 20 ng/mL of plasma (six replicates each), and each time the internal standards were added to the plasma with the same concentration. After UHPLC–MS/MS analysis, the relative recoveries were calculated from the ratio of the signal intensity of QCs extracted by HF–LPME versus the signal intensity of QCs in the samples. The recoveries for the three compounds varied from 92.1% to 106.0% with the RSD lower than 15%. 3.2.6. Stability Stability of the analytes in plasma was verified by QC samples at 4 ◦ C for 24 h, at −20 ◦ C for 3 months and after three freeze/thaw cycles. The stabilities of BP, NBP and NLX in human plasma were from −12.50% to 14.67%. 3.3. Comparison between the method HF–LPME and SPE This paper compared the pretreatment method of HF–LPME with SPE to prove the reliability of this method. As the paper reported method of SPE with the BP, NBP and NLX in human plasma [29], using the Oasis® MCX cartridge (Waters), each sample was

added with methanol to deposit protein, and then with hydrochloric acid to make the solution acidity. After ultrasonic mixing, the sample was centrifuged at 4 ◦ C for 5 min at 12,000 r/min. The upper part of the sample was transferred to SPE cartridge, which was previously conditioned with 1 mL of methanol and 1 mL of HCl. Then SPE cartridge was washed by 1 mL 5% ammonia in methanol and dried for 1 min under vacuum condition. The elution was evaporated to dryness with N2 , and reconstituted with 50 ␮L of the mobile phase. After ultrasonic mixing for 5 min, analyses were analyzed by UHPLC–MS/MS. Table 3 compared two methods for the BP, NBP and NLX by RSD in different concentration levels (0.25, 2.5 and 20 ng/mL). It could be found that the RSD and accuracy of HF–LPME were as well as SPE. Table 4 gave a comparison about expenditure, organic solvent volume, signal intensity. As can be seen the HF–LPME has some advantages: cheap, convenient, steady and less pollution. 3.4. Pharmacokinetic experiment and pharmacokinetic parameters The optimized method was applied to determine the concentration of BP, NBP and NLX in the plasma samples collected from some healthy volunteers submitted to single dose treatment with BP and NLX sublingual (4:1) by two dose groups: group A (4 mg, 3.2 mg of BP and 0.8 mg of NLX) and group B (16 mg, 12.8 mg of BP and 3.2 mg of NLX). Sampling time: before take pills and after take pills 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 36, 48, 60 and 72 h. Forearm venous blood collected 5 mL, kept 30 min, centrifuged for 20 min at 4000 r/m, then saved at −20 ◦ C. The plasma samples were analyzed in this method, then we got the concentration-versus-time profile of BP, NBP and NLX in plasma. The mean plasma concentration-versus-time profiles of BP, NBP and NLX were shown in Fig. 4. The plasma concentration of three targets peaked around 1 h after administration, with a Cmax

Table 5 The main pharmacokinetic parameters of BP, NBP and NLX (n = 6). Compound

Cmax (ng/mL) Tmax (h) T1/2 (h) AUC (h ng/mL)




Group A*

Group B*

Group A

Group B

Group A

Group B

2.33 ± 0.15 1.75 ± 0.25 16.05 ± 1.35 20.09 ± 4.53

7.31 ± 0.50 1.50 ± 0.35 21.03 ± 3.23 76.99 ± 8.09

0.33 ± 0.50 2.25 ± 0.75 16.2 ± 1.88 4.60 ± 0.50

4.28 ± 0.47 1.25 ± 0.25 19.8 ± 2.70 73.12 ± 3.14

0.23 ± 0.04 1.13 ± 0.38 1.04 ± 0.19 0.48 ± 0.16

0.68 ± 0.03 1.25 ± 0.25 1.51 ± 0.21 1.64 ± 0.18

*Group A: 4 mg (BP/NLX = 1/4); *Group B: 16 mg (BP/NLX = 1/4).

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4. 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 plasma samples. This method presented a high enrichment and good sensitivity, while enabled efficient sample clean-up. Application of HF–LPME–UHPLC–MS/MS for analytes in plasma samples was successful, and it was contributed to pharmacokinetic studies of BP, NBP and NLX. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at 2014.01.029. References [1] [2] [3] [4] [5] [6]

[7] [8] [9] [10] [11] [12]

[13] [14] [15] [16] [17] [18] [19] Fig. 4. Plasma concentration-versus-time profile of BP, NBP and NLX.

of 6.4 ng/mL for BP, 4.2 ng/mL for NBP and 0.6 ng/mL for NLX. Application of HF–LPME–UHPLC–MS/MS for analysis of BP, NBP and NLX in plasma samples was successful. We calculated pharmacokinetic parameters by the software of Kinetica 4.4, through the two groups of dose of BP, NBP and NLX in plasma samples. The main pharmacokinetic parameters of BP, NBP and NLX were shown in Table 5. As can be seen from the experimental results, the Tmax of NLX was 1.25 h which smaller than BP. The results showed the rapid absorption of NLX, and it is helpful for clinical treatments.

[20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30]

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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.

A hollow fiber liquid phase microextraction (HF-LPME) combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS...
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