Reduced graphene oxide as an efficient sorbent in microextraction by packed sorbent: Determination of local anesthetics in human plasma and saliva samples utilizing liquid chromatography-tandem mass spectrometry

Accepted Manuscript Reduced graphene oxide as an efficient sorbent in microextraction by packed sorbent: Determination of local anesthetics in human plasma and saliva samples utilizing liquid chromatographytandem mass spectrometry

Mazaher Ahmadi, Mohammad Mahdi Moein, Tayyebeh Madrakian, Abbas Afkhami, Soleiman Bahar, Mohamed AbdelRehim PII: DOI: Reference:

S1570-0232(18)30846-8 doi:10.1016/j.jchromb.2018.07.036 CHROMB 21309

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

3 June 2018 22 July 2018 26 July 2018

Please cite this article as: Mazaher Ahmadi, Mohammad Mahdi Moein, Tayyebeh Madrakian, Abbas Afkhami, Soleiman Bahar, Mohamed Abdel-Rehim , Reduced graphene oxide as an efficient sorbent in microextraction by packed sorbent: Determination of local anesthetics in human plasma and saliva samples utilizing liquid chromatography-tandem mass spectrometry. Chromb (2018), doi:10.1016/ j.jchromb.2018.07.036

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ACCEPTED MANUSCRIPT Reduced graphene oxide as an efficient sorbent in microextraction by packed sorbent: determination of local anesthetics in human plasma and saliva samples utilizing liquid chromatography-tandem mass spectrometry

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Mazaher Ahmadi1, Mohammad Mahdi Moein2, Tayyebeh Madrakian1, Abbas Afkhami1,

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Soleiman Bahar3, Mohamed Abdel-Rehim2,4* Faculty of Chemistry, Bu-Ali Sina University, Hamedan, Iran.

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Department of Clinical Neuroscience, Center for Psychiatry Research, Karolinska Institutet and

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1

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Department of Chemistry, Faculty of Science, University of Kurdistan, Sanandaj, Iran.

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Stockholm County Council, SE-171 76 Stockholm, Sweden.

Division of Materials and Nanofysik (MNF), KTH Royal Institute of Technology, Stockholm,

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*Corresponding author:

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Sweden.

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Prof. Mohamed Abdel-Rehim, Tel: +46707108122

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Email: [email protected]; [email protected]

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ACCEPTED MANUSCRIPT Abstract Herein, reduced graphene oxide (RGO) has been utilized as an efficient sorbent in microextraction by packed sorbent (MEPS). The combination of MEPS and liquid chromatography-tandem mass spectrometry has been used to develop a method for the extraction

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and determination of three local anesthetics (i.e. lidocaine, prilocaine, and ropivacaine) in human

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plasma and saliva samples. The results showed that the utilization of RGO in MEPS could

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minimize the matrix effect so that no interfering peaks at the retention times of the analytes or internal standard was observed. The high extraction efficiency of this method was approved by

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mean recoveries of 97.26-106.83% and 95.21-105.83% for the studied analytes in plasma and

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saliva samples, respectively. Intra- and inter-day accuracies and precisions for all analytes were in good accordance with the international regulations. The accuracy values (as percentage

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deviation from the nominal value) of the quality control samples were between -2.1 to 13.9 for

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lidocaine, -4.2 to 11.0 for prilocaine and between -4.5 to -2.4 for ropivacaine in plasma samples while the values were ranged from -4.6 to 1.6 for lidocaine, from -4.2 to 15.5 for prilocaine and

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from -3.3 to -2.3 for ropivacaine in human saliva samples. Lower and upper limit of

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quantification (LLOQ, ULOQ) were set at 5 and 2000 nmol L-1 for all of the studied drugs. The correlation coefficients values were ≥0.995. The limit of detection values were obtained 4 nmol

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L-1 for lidocaine and prilocaine, and 2 nmol L-1 for ropivacaine.

Keywords: Local anesthetics; Microextraction by Packed Sorbent; Reduced Graphene Oxide; LC-MS/MS; Plasma; Saliva.

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ACCEPTED MANUSCRIPT 1. Introduction The availability of certain analytical instruments such as chromatographic systems has always been a determinative issue in what sample preparation method should be utilized. In case of complex matrixes such as biological fluids, even chromatographic methods which provide

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high separation efficiency, cannot be directly used [1]. Therefore, one or more pretreatment steps

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with an efficient sample preparation technique are necessary to enhance the instrument efficiency and lifetime. Sample preparation techniques can increase sample enrichment and signal

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enhancement by elimination of the interferences through the extraction of the analytes of interest into a liquid solvent or a solid phase [1]. In recent year, there have been many sample

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preparation techniques utilizing solid phases such as solid phase extraction and microextraction

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techniques, stir-bars, stir-cake, rotating-disk extraction techniques, and dispersive extraction methods [2-9]. Amongst them, microextraction by packed sorbent (MEPS) has more

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compatibility with the chromatographic systems which was originally designed for liquid

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chromatography instruments [10]. In MEPS, the sorbent is packed inside a micro-syringe in

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different positions such as the barrel, needle, or BIN space [1]. The limited adsorption capacity of the available adsorbents could lead to low recoveries of

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the analyte of interest which consequently decreases the overall sensitivity of the utilized detection method. To address this issue, more selective adsorbents of higher adsorption capacities can be utilized. These adsorbents usually are in nano-scale dimensions [11, 12]. Nanomaterials present different properties compared to bulk materials. One of these different properties which can improve the efficiency of the available adsorbents is the higher surface area to volume ratio of nanomaterials compared to bulk materials [13, 14]. Unfortunately, there are few reports on utilization of nanomaterials for bioanalysis in some extraction based techniques 3

ACCEPTED MANUSCRIPT such as MEPS [11]. Since MEPS was invented by Abdel-Rehim [15], various sorts of materials have been utilized as the sorbent in MEPS including most frequently silica C8 and C18, polystyrene, polydivinylbenzene, and molecularly imprinted polymers [16-21]. In the current paper, reduced graphene oxide (RGO) has been utilized as the sorbent of MEPS for the

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extraction of local anesthetics for the first time. RGO, highly reduced graphene oxide, provides

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relatively high adsorption capacity toward aromatic compounds while it is cost-effective and

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widely accessible [22-24].

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Local anesthetics are a class of drugs which are widely used to relieve pain during and after medical procedures [25]. These drugs interrupt neural conduction process involved in pain

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feeling by inhibiting sodium ions uptake and can reversibly block nerve function [26-28].

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Although, local anesthetics are relatively free of side effects, accidental intravascular injection or overdose usage can cause systemic and localized toxicity in addition to some adverse effects

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such as allergic reactions [29]. In addition some metabolites of these drugs such as 2,6-xylide

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and 4-OH-2,6-xylidene (major of lidocaine metabolites) and o-Toluidine (major of prilocaine metabolites) have been shown to be carcinogenic and therefore, determination of local anesthetics

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and their metabolites are of interest. In this regard, some sample preparation techniques such as

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solid phase extraction [28, 30, 31], solid phase microextraction [32], and MEPS [26, 33] have been utilized to extract the drugs from biological samples prior to their introduction to the detection instruments. However, to our best knowledge, there is no previous report on the application of RGO as the sorbent of MEPS for the extraction of local anesthetics in biological matrixes such as plasma and saliva. In this paper, for the first time, RGO has been utilized as an effective adsorbent in MEPS for the extraction of three local anesthetics (i.e. lidocaine, prilocaine, and ropivacaine) in human 4

ACCEPTED MANUSCRIPT plasma and saliva samples prior to liquid chromatography-tandem mass spectrometry (LCMS/MS) measurement. Various parameters which could potentially affect the extraction efficiency have been optimized and, under the optimized conditions, a gradient liquid chromatographic method was developed and fully validated according to the international

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guidelines.

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2. Experimental 2.1.Reagents and Materials

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Lidocaine, prilocaine, and ropivacaine were obtained from Department of Medicinal Chemistry, AstraZeneca (Södertälje, Sweden) as hydrochlorides. Pentycaine also from

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AstraZeneca was used as the internal standard (I.S.). The chemical structure of the investigated

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analytes is shown in Fig. 1. HPLC-grade solvents (acetonitrile, methanol, water) from Merck (Darmstadt, Germany) were used for LC-MS/MS measurements. Ammonium acetate and formic

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acid with the highest available purity were obtained from Merck. RGO (BET surface area 450 m2

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g-1) from Aldrich (St. Louis, Missouri, United States) was used as the sorbent of MEPS. Nitrogen gas and high-purity argon gas as the drying and nebulizing gasses, and the collision gas,

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respectively, were purchased from AGA (Stockholm, Sweden).

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The stock solutions were prepared in deionized water and kept in dark at refrigerator (28ºC). The stock solution of the internal standard was prepared in methanol and the stability of the stock solutions was found to be about at least 6 months at 8ºC. The standard solutions of the concentration of interest were daily prepared by appropriate dilution of the stock solutions with deionized water and then into plasma or saliva to achieve the required concentration. For the validation experiments, the quality control (QC) and calibration samples were prepared in blank plasma and saliva samples. Blank human plasma samples containing sodium heparin as the anti5

ACCEPTED MANUSCRIPT coagulant were provided by Karolinska University Hospital, Stockholm, Sweden. Saliva samples were collected from healthy donors (n=6). The calibration samples (5, 10, 25, 100, 500, 1000, and 2000 nmol L-1) and QC samples of low (15 nmol L-1), medium (750 nmol L-1), and high (1600 nmol L-1) levels in plasma and saliva were prepared daily as mentioned above.

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Fig. 1.

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2.2.LC-MS/MS measurements

A liquid chromatography instrument with a Quattro-micro mass spectrometer detector

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(Waters, Manchester, UK) equipped with an electrospray ionization source (ESI) was utilized to

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investigate the analytes levels in the eluate of MEPS unit. The LC was equipped with two LC10Advp pumps from Shimadzu (Kyoto, Japan), an autosampler (CTC-Pal, Analytics AG,

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Zwingen, Switzerland), a 50 mL sample loop, and a 4.6 mm i.d.×100 mm length YMC-Pack

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ODS-A column (particle size: 3 µm) from YMC Europe GmbH (Schermbeck, Germany). For the LC separation, a gradient mobile phase program was used using (A) formic acid

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(0.1%) in ammonium acetate (0.1 mol L-1)-acetonitrile (80:20, %v/v), and (B) formic acid (0.1%)

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in ammonium acetate (0.1 mol L-1)-acetonitrile (20:80, %v/v) by starting from mobile phase B from 0.5% to 80% of mobile phase A within 8 minutes with a pause (isocratic) at 80% for 2

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minutes. Finally, at 10.1 min, the mobile phase B was set at 0.5% again. The flow rate was 300 µL min-1. The injected sample volume was 30 µL. For the MS/MS detection, the mass analyzer was operated at positive ion mode using MassLynx software (version 4.1). The instrumental parameters were: the MS source block temperature and desolvation temperature: 150 and 400˚C, respectively; the capillary, cone and extractor voltages: 4 kV, 25 V, and 2.0 V, respectively; the collision energy: 20 eV. The

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ACCEPTED MANUSCRIPT analytical signal was peak area ratios the analyte of interest to the I.S. Multiple reaction monitoring (MRM) scan mode using the precursor ions at m/z (M + 1) (m/z 235, 221, 275, 303), and the product ions at m/z 86, 86, 126 and 154 for quantification of lidocaine, prilocaine, ropivacaine, and pentycaine (I.S.), respectively, were utilized.

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2.3.Sample Pretreatment and MEPS Procedure

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For MEPS experiments, 250 µL gas-tight syringes (Hamilton-Bonaduz, Schweiz) were

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used. RGO (2 mg) was packed inside the barrel of the syringe as a plug and was tightened between two filters.

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The plasma and saliva samples were carefully homogenized after thawing using a vortex

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mixer. Then, the homogenized samples were spiked with the standard working solutions. A 200 µL of the spiked samples were diluted with water (200 µL) followed by adding 100 µL I.S. in

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methanol (20 ng mL-1, the methanol was used to help in protein precipitation). Then, the mixture

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was centrifuged (3000 rpm) at 25 ºC for 15 min. After separating the precipitated proteins, the supernatant fluid was introduced to the MEPS procedure (Table 1). After applying the MEPS

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procedure, the eluate (30 μL) was introduced to LC-MS/MS automatically. To reutilize the

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MEPS sorbent (>100 times) and to remove the macromolecules and memory effect, the sorbent

extraction.

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was washed consecutively with the eluent (4×100 µL) and water (4×200 µL) after each

Table 1

2.4.Method Validation

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ACCEPTED MANUSCRIPT Food and Drug Administration guidelines were obeyed in the method validation experiments [34, 35]. The precision (%RSD) and accuracy (percentage deviation) of the method were determined by using the QC samples (n = 5) of high, medium, and low concentrations. The inter-day precision and accuracy were evaluated in three consecutive days. In the case of

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accuracy, when back-calculated concentrations were within ±15% of the nominal value, except

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for the lower limit of quantification (LLOQ) which ±20% was considered, the data was accepted.

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QC samples outside ±30% from the mean value were regarded as outliers and were not included in the calculations. In the case of precision, the precision values will be accepted if not exceeding

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15% (20% for LLOQ).

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The peak area ratio of the investigated drugs to the I.S. versus the concentration of the drugs, were used to obtain the calibration. At least 75% or a minimum of six standards when

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back-calculated concentrations including the upper limit of quantification (ULOQ) were within

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±15%, except for the lower limit of quantification (LLOQ) which was within ±20% of the nominal value. Values falling outside these limits were excluded, provided they did not change

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the established model. In the case of precision, RSD values ≤ 15 were accepted (except for

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LLOQ of which 20% was considered). To evaluate the matrix effect, the peak response of the post-extracted spiked samples at QC

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levels were compared with those of the pure standards containing equivalent amounts of the analytes prepared in methanol and formic acid (90:10, %v/v). To evaluate the method selectivity, six different blank human plasma and saliva samples without the I.S. were applied to the developed method to confirm interference-free at the peaks regions. 3. Results and Discussion 3.1. Method Development 8

ACCEPTED MANUSCRIPT Various parameters affect the extraction efficiency such as sorbent conditioning solutions, the number of the MEPS extraction cycles, sample volume, sample pH and the composition of washing and elution solutions were investigated. To maximize MEPS extraction recoveries, different adsorption and desorption parameters were optimized. Sorbent preconditioning is the

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first step of the MEPS process. Usually, the sorbent pre-conditioning is performed with a suitable

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organic solvent followed by washing with deionized water [10]. In this study, the

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preconditioning was performed with 200 μL methanol followed by washing with 200 μL deionized water. In the next step, the sample can be drawn inside the syringe to interact with the

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sorbent. It can be done by two modes: draw-eject in the same vial or draw and eject into waste

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[16]. In this work, draw-eject in the same vial was chosen to have the highest extraction efficiencies. The number of cycles was also investigated, and it was seen that 6 cycles could

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provide quantitative extractions (removal efficiency > 96%).

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The adsorption process should be followed by a desorption step utilizing a proper eluent. An ideal eluent should quantitatively desorb the adsorbed analytes. In case of mass spectrometric

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measurements, the eluent should also be compatible with mass spectrometry interface. Therefore,

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in this study, the different volume ratio of formic acid in methanol (0-12%) were evaluated as the eluent. The result showed that 10% formic acid in methanol (2×100 μL) could act as the most

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efficient eluent to quantitatively desorb the adsorbed drug molecules. In order to eliminate carryover, the sorbent was washed with the eluent (3×200 µL) and water (3×200 µL) consecutively after each extraction. The results showed that these post-washing steps are sufficient to quantitatively eliminate the carry-over (<0.01%). 3.2.Method Validation 3.2.1. Matrix Effect and Selectivity

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ACCEPTED MANUSCRIPT To evaluate the matrix effect, the peak area of the post-extracted plasma and saliva samples at QC concentration levels were compared with those of the pure standards containing equivalent amounts of the analytes. The peak area ratios (post-extracted/pure standards) values were 94.397.7% and 95.6-98.1% for plasma and saliva, respectively. The results indicated that there is no

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matrix effect since no significant ion suppression or enhancement was present.

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Figure 2 shows, the blank plasma and saliva samples chromatograms after treatment with

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the developed method. As it can be seen, at the retention times of the investigated analytes as well as the I.S. (Fig. 3) no significant interferences were detected indicating high selectivity of

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the developed method towards the investigated local anesthetics.

Fig. 2

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Fig. 3

3.2.2. Linearity

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The calibration curves using seven standard concentrations excluding zero concentration in

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plasma or saliva using linear regression equation (the weighting factor, 1/x) were constructed for each drug. Table 2 shows the regression parameters for each calibration curve. LLOQ and

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ULOQ were set to 5 and 2000 nmol L-1; respectively. The correlation coefficients were more than 0.995. The LOD was 4 nmol L-1 for lidocaine and 2 nmol L-1 för prilocaine, and ropivacaine. Plasma calibration curves showed higher standard deviation values in terms of slopes and intercept (Table 2) and this is due to the plasma matrix complexity compared to saliva.

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Table 2

3.2.3. Accuracy and Precision

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Table 3 shows the accuracy (expressed as percentage deviation) and precision (expressed

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as percentage RSD) of the QC samples determination using the developed method. The obtained

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intra-day mean accuracies were found to be -2.74-6.83% and -4.79-5.83% in plasma and saliva, respectively, displaying that acceptable high accuracies were achieved. The RSD values were

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2.39-8.76% and 5.69-14.82% in plasma and saliva, respectively, indicating the acceptable

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precision of the method. The mean recoveries were 97.26-106.83% and 95.21-105.83% in

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plasma and saliva, respectively, showing high extraction efficiency of the developed method.

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Table 3

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4. Conclusions

Lidocaine, prilocaine, and ropivacaine were extracted from plasma and saliva utilizing

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MEPS followed by gradient liquid chromatography with mass spectrometric detection. RGO was successfully used as the sorbent for MEPS and it showed good extraction capacity and good selectivity for the extraction of extraction lidocaine, prilocaine, and ropivacaine in plasma and saliva samples. The method was fully validated in terms of linearity, selectivity, accuracy, and precision. The method provides a good selectivity, high sensitivity for the determination of the investigated drugs in human plasma and saliva samples. Furthermore, the matrix effect and

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ACCEPTED MANUSCRIPT carry-over were negligible. The method is accurate and can be used for the quantification of the investigated drugs in plasma and saliva samples.

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ACCEPTED MANUSCRIPT Table 1. The optimized conditions for the MEPS procedure. The optimized parameters

Conditioning

Methanol (200 μL) and water (200 μL) consecutively

Extraction

Draw-eject in the same vial, 6×200 μL

Washing solution

Water: methanol (95:5, %v/v), 200 μL

Elution

Methanol: formic acid (90:10, %v/v), 2×100 μL

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MEPS procedure step

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ACCEPTED MANUSCRIPT Table 2. The analytical parameters of the developed method for the determination of the studied drugs in plasma and saliva samples (n=3). Parameter

Plasma

Saliva

Prilocaine

Ropivacaine

Lidocaine

Prilocaine

Ropivacaine

Linearity range (nmol L-1)

5-2000

5-2000

5-2000

5-2000

5-2000

5-2000

Slope

0.030±0.011

0.015±0.004

0.186±0.073

0.041±0.003

0.017±0.002

0.207±0.046

Intercept

0.292±0.170

0.060±0.030

3.155±0.982

0.104±0.032

0.044±0.001

1.389±0.512

Correlation coefficient

0.998±0.001

0.998±0.001

0.999±0.000

0.995±0.005

0.999±0.001

0.998±0.002

LOQ (nmol L-1)

4

4

2

4

4

2

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Lidocaine

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ACCEPTED MANUSCRIPT Table 3. Accuracy and precision of the developed method for the determination of the studied drugs in human plasma and saliva samples. Matrix

QC level

Drug

Intra-day (n=5)

Inter-day (n=15)

(nmol L-1) Mean recovery

RSD

Deviation

(%)

(%)

RSD

Deviation

(%)

(%)

113.94 97.92 103.60

17.41 11.08 3.81

13.94 -2.08 3.60

Mean recovery (%)

Plasma

IP

T

(%)

15 750 1600

102.50 98.56 104.69

7.85 2.39 6.69

2.50 -1.44 4.69

Prilocaine

15 750 1600

106.83 97.36 97.26

4.43 5.69 3.12

6.83 -2.64 -2.74

111.00 95.82 96.72

19.14 4.91 11.54

11.00 -4.18 -3.28

Ropivacaine

15 750 1600

98.17 98.35 99.79

5.04 8.76 6.99

-1.83 -1.65 -0.21

95.89 95.25 97.26

4.02 7.11 8.00

-4.11 -4.75 -2.74

Lidocaine

15 750 1600

97.33 97.89 105.83

14.82 12.57 8.64

-2.67 -2.11 5.83

95.42 101.55 100.52

18.21 10.30 6.01

-4.58 1.55 0.52

Prilocaine

15 750 1600

105.50 96.76 95.21

5.69 9.71 7.56

5.50 -3.24 -4.79

115.50 96.84 95.31

16.24 9.84 10.80

15.50 -3.16 -4.69

Ropivacaine

15 750 1600

105.50 99.38 101.40

6.57 6.13 8.78

5.50 -0.62 1.40

97.67 96.67 97.64

4.84 4.31 9.32

-2.33 -3.33 -2.36

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CR

Lidocaine

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ACCEPTED MANUSCRIPT Figures Captions: Fig. 1. The chemical structures of the investigated drugs and the internal standard. Fig. 2. MRM transitions obtained for blank plasma and saliva samples after extraction using the developed MEPS method.

IP

T

Fig. 3. MRM transitions obtained for spiked (25 nmol L-1) plasma (A) and saliva (B) samples

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after the extraction using the developed MEPS method.

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ACCEPTED MANUSCRIPT FIGURES

H N

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H N

N

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Ropivacaine

US

Lidocaine

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AN

N H

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NH

ED

Prilocaine

H N N O

Pentycaine (internal standard)

AC

CE

PT

Fig. 1.

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ACCEPTED MANUSCRIPT

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Fig. 2.

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ACCEPTED MANUSCRIPT

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Fig. 3.

ACCEPTED MANUSCRIPT Highlights - Use of reduced graphene oxide as sorbent for MEPS technique. - The quantification of local anesthetics in plasma and saliva samples was carried out - The bioanalytical method was validated.

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- The method showed good selectivity, good accuracy and precision.

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