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Analysis of four antidepressants in plasma and urine by gas chromatography-mass spectrometry combined with sensitive and selective derivatization
Accepted Manuscript Title: Analysis of Four Antidepressants in Plasma and Urine by Gas Chromatography-Mass Spectrometry Combined with Sensitive and Selective Derivatization Authors: Yanru Feng, Min Zheng, Xue Zhang, Kai Kang, Weijun Kang, Jie Yang, Kaoqi Lian PII: DOI: Reference:
S0021-9673(19)30406-6 https://doi.org/10.1016/j.chroma.2019.04.038 CHROMA 360184
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
10 January 2019 11 April 2019 13 April 2019
Please cite this article as: Feng Y, Zheng M, Zhang X, Kang K, Kang W, Yang J, Lian K, Analysis of Four Antidepressants in Plasma and Urine by Gas ChromatographyMass Spectrometry Combined with Sensitive and Selective Derivatization, Journal of Chromatography A (2019), https://doi.org/10.1016/j.chroma.2019.04.038 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Analysis of Four Antidepressants in Plasma and Urine by Gas Chromatography-Mass Spectrometry Combined with Sensitive and Selective Derivatization
c,
**,
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Yanru Feng a, Min Zheng a, Xue Zhang a, Kai Kang b, Weijun Kang a, Jie Yang
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Kaoqi Liana, d, *
School of Public Health, Hebei Medical University, Shijiazhuang, 050017, China
b
School of Pharmacy, Hebei Medical University, Shijiazhuang, 050017, China
c
The pharmaceutical department of the Third Hospital of Hebei Medical University,
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a
Hebei Key Laboratory of Environment and Human Health, Shijiazhuang 050017,
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d
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Shijiazhuang 050051, China
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China
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*Corresponding author:
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Department of Sanitary Inspection, School of Public Health, Hebei Medical University, Shijiazhuang, P. R. China. E-mail addresses: [email protected] (K.Q. Lian)
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** Corresponding author: The pharmaceutical department of the Third Hospital of Hebei Medical University Shijiazhuang, P. R. China.E-mail addresses: [email protected] (J. Yang)
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Highlights
A simple and fast derivatization method was used to analysis of four ATDs by GC-MS.
The method also provided excellent sensitivity and selective without side
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product. The LOQs of the analytes were significantly lower than actual sample
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concentration.
The method will provide a new strategy for the detection of ATDs in plasma
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and urine.
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ABSTRACT
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A sensitive and selective method was developed for simultaneous determination
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of four antidepressants (ATDs) in plasma and urine samples by gas chromatographymass spectrometry (GC-MS) based on an N-nitrosation reaction. In this study,
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fluoxetine (Flu), nortriptyline (Nor), maprotiline (Map), and paroxetine (Paro) were
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first derivatized with sodium nitrite to appropriate N-nitrosamines under acidic condition, then the derivatives were easier to detect by GC-MS. The derivatization conditions including the amount of hydrochloric acid, the amount of sodium nitrite,
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reaction temperature, reaction time and the extraction reagents were optimized. Under the optimal conditions, the limit of detections (LODs) and limit of quantitations (LOQs) were in the range of 0.04-1.38 μg L-1 and 0.14-4.62 μg L-1, respectively. Low, medium, and high concentrations of antidepressants were added in plasma and urine 2
samples, spiked recovery ranged from 85.88% to 110.34% for plasma and 80.64% to 113.07% for urine, respectively. The derivatization reaction was very quickly, only 5 min was needed for the reaction process, in addition, the proposed method exhibited superior sensitivity and selectivity, it showed sufficient advantages for determination
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of Flu, Nor, Map, and Paro in plasma and urine of patients.
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Keywords: antidepressants, derivatization, Gas chromatography-mass spectrometry,
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N-Nitrosamine, plasma, urine
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1. Introduction
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Depression is one of the most common chronic or recurrent mental illnesses in
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modern society. It is often characterized by inattention, reduced self-confidence,
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pessimism, guilt, sleep disturbance, self-harm, and ultimately suicide [1, 2]. antidepressants (ATDs) are widely used to alleviate and treat symptoms of mental
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illness. Due to their side effects, toxicity and severe drug interactions [3],
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first-generation antidepressants such as tricyclic antidepressants (TCAs) are gradually being replaced by a new generation of ATDs, such as selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) [4].
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However, due to the narrow safety window of antidepressant drugs and the large patient variation, patients taking medication need long-term monitoring of vivo concentration [5, 6]. At the same time, due to the poor compliance of patients with depression, patients often suffer from poisoning due to overdose or misuse [7]. 3
Therefore, the establishment of sensitive, rapid, and reliable analytical detection techniques for ATDs is of great significance for identifying drug poisoning, monitoring therapeutical drugs, assisting clinicians to adjust drug dosage, and achieving individualized rational use of medications [8, 9]. performance
liquid
chromatography
(HPLC)
[10,
11],
liquid
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High
chromatography-mass spectrometry (LC-MS) [12-14], and capillary electrophoresis
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(CE) [15, 16] have been used for the analysis and detection of ATDs. The biological samples involved are mainly plasma, urine and whole blood. Due to the complex
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interference of the biological sample matrix, separation and enrichment techniques are
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needed for processing. The previously reported sample pretreatment methods are
phase
micro-extraction
(SPME)
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solid
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liquid-liquid extraction (LLE) [17, 18], solid phase extraction (SPE) [16, 19, 20], [10,
21],
dispersive
liquid-liquid
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microextraction (DLLME) [22, 23]; liquid phase microextraction (LPME) [24], ionic liquid-liquid-liquid microextraction (IL-LLME) [4]; stir bar sorptive extraction (SBSE)
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[25, 26], single-drop microextraction/microvolume pipette extraction (SDME/MVPE)
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[15], and the others [4, 27-31], which have the disadvantages of troublesome operation, lengthy procedure , high cost, disposable, and high consumption of organic
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solvents.
Gas chromatography (GC) is a commonly used method in mixture analysis. It
has the characteristics and advantages of mature technology, easy to master, high detection sensitivity, high separation efficiency, high selectivity, low detection limit, low sample consumption, rapidness, and convenience. However, ideal compounds for 4
GC analysis should have low boiling points, good thermal stability, and low molecular polarities. In order to convert compounds that are either undetectable directly by GC or detected with a low sensitivity to those ideal ones that are easily detected by GC analysis, chemical derivatization techniques are commonly used, including acylation
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[22, 32], silanization [33], and esterification [23]. Acylation is the conversion of a compound containing active hydrogen into an ester, sulfide, or amide by means of a
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carboxylic acid; silanization is generally the replacement of active hydrogen by
silylation; esterification is the reaction of alcohols with carboxylic acids to form esters.
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There are several reports on the application of GC in combination with derivatization
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technology to ATDs. However, these derivatization methods have the problems of
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relatively complicated operation procedures, long reaction times, and numerous side
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reactions.
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The authors previously established an innovative new method for the detection of ketamine in urine and plasma with gas chromatography-mass spectrometry
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(GC-MS) by using the reaction of secondary amines and sodium nitrite to form
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N-nitrosamine compounds under acidic conditions [34]. This derivatization method has three major advantages. First, the product N-nitrosamines are fat-soluble substances, making them readily extracted by organic solvents; being less polar, the
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products are very suitable for GC-MS detection with greatly improved detection sensitivity. Second, since only secondary amine molecules can be quantitatively reacted in this reaction, a large amount of matrix interference can be eliminated, greatly improving selectivity. Last, this reaction is fast, shortening the analysis cycle. 5
Table 1
Many ATDs such as fluoxetine (Flu), nortriptyline (Nor), maprotiline (Map), and
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paroxetine (Paro) were found to contain secondary amine groups, the chemical structure, some physicochemical properties (pKa, log P) of Flu, Nor, Map, and Paro
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were shown in Table 1 [35, 36]. Previous studies have found that the sensitivity of direct extraction from biological sample followed by GC-MS detection was very low.
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Theoretically, such ATDs with secondary amines can react with sodium nitrite under
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acidic conditions to form corresponding N-nitrosamine. However, application of this
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derivatization approach with gas chromatography or gas chromatography-mass
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spectrometry analysis of ATDs has not been reported. Through optimization of the
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reaction conditions, extraction parameters, and GC-MS separation and analysis procedures, we have established a high-sensitivity and high-selectivity GC-MS
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approach for the detection of Flu, Nor, Map, and Paro in plasma and urine. This
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method may be applied to the detection of similar clinical drug content and side effect
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control, and play a positive role in assisting clinicians to adjust the drug dosage.
2. Experimental 2.1. Chemicals and reagents Nortriptyline hydrochloride (Nor) was purchased from Sigma (St. Louis, MO, USA), Fluoxetine hydrochloride (Flu), Maprotiline hydrochloride (Map) and 6
Paroxetine hydrochloride (Paro) were bought from National Institutes for Food and Drug control (Beijing, China). Analytical reagent grade sodium nitrite (NaNO2) and hydrochloric acid (HCl) were from Hubei university chemical factory (Hubei, China) or Shijiazhuang Huadi chemical industry and trade co. LTD. (Shijiazhuang, China)
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were used as derivatizing reagent. Analytical grade chloroform, dichloromethane, carbon disulfide obtained from Xilong Chemical Factory (Shantou, China) or Tianjin
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General Chemical Reagent Factory (Tianjin, China) and HPLC grade n-hexane, ethyl acetate, toluene supplied by Tianjin commie chemical reagent co. LTD. (Tianjin,
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China) or Li’anlongbohua pharmaceutical chemical co. LTD (Tianjin, China). were
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tested as extraction solvents. Methanol (MeOH, HPLC grade) was supplied by
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Oceanpakalexative chemical., Ltd (Gothenburg, Sweden). Ultra-pure water was
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obtained from Sichuan Youpu super pure technology co. LTD. UPR-II-20L (Sichuan,
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China).
2.2. Preparation of standard and working solutions
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Primary stock solutions of each individual drug (Flu, Nor, Map, and Paro) were
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prepared in MeOH at a concentration of 1 mg ml-1 and stored light protected at -20℃. Standards working solutions of the mixtures of each compound at 50, 50, 125 and 250 μg L-1 (Flu, Nor, Map, and Paro) respectively were prepared by proper dilutions with
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ultrapure water.
2.3. Sample Preparation Human blank plasma and urine were stored at -20 °C for the preparation of quality control (QC) samples. Three concentrations of QC samples were prepared in 7
plasma and urine (low, medium, and high): 2, 20, and 65 μg L-1 for Flu and Nor, 5, 50, and 162.5 μg L-1 for Map, and 10, 100, and 325 μg L-1 for Paro, respectively. The sample solutions were filtered through a 0.45 μm filter before derivatization. 2.4. Derivatization
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Into a 10.0 mL open glass tube, 2.0 mL of standard solution or sample solution was added followed by the addition of 100 μL of 1.0 mol L-1 HCl, vortexed prior to
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the addition of 50 μL of saturated sodium nitrite solution dropwise under vortexing.
The sample tube was then sealed and heated at 60 °C for 5 min to carry out the
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derivatization reaction. After cooling, 2.0 mL of dichloromethane was added for
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extraction. The mixture was vortexed for 1.5 min, allowed to stand for 5 min for
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stratification (plasma and urine samples were centrifuged at 15000 rpm for 5 min).
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With a micropipette, 1.2 mL of the organic phase was transferred into a glass tube
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with a stopper, and blow-dried with nitrogen at 50 °C. The residue was dissolved in 50 μL of dichloromethane and placed in an ice bath to prevent dichloromethane
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volatilization, 1 μL of which was taken for GC-MS analysis.
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2.5. GC-MS analysis
GC-MS analysis was performed on an Agilent (Little Falls, DE, USA) gas
chromatograph 7890A equipped with an electronically controlled split/splitless
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injection port, an inert 5975C mass selective detector with electron impact (EI) ionization chamber, and a 7683B Series injector/autosampler. Chromatographic separation was conducted with an HP-5 MS 30 m×0.25 mm I.D., 0.25 μm film thickness column (Agilent, CA, USA).GC oven programmed 8
temperature condition was shown in Table 2.Injections (1μL) were done at 250 °C in the split mode, split ratio was 5:1. Helium was used as the carrier gas with constant flow rate at 1.2 mL min-1. The mass spectrometer (MS) was operated at electronic impact (EI, 70 eV) mode, ion source temperature at 230 °C, and MS quadrupole
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temperature at 150 °C. The MS transfer line temperature was held at 280 °C. In the full-scan mode, preliminary mass spectra of the target analytes were obtained from
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m/z 0 to 750 AMU. Selected-ion monitoring mode used for the selectivity and sensitivity concerns, the monitored ions were selected as follows: Flu, m/z: 104.1,
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117.1, 177.1; Nor, m/z: 204.1, 219.1, 232.1; Map, m/z: 178.1, 203.1,248.2, 276.1;
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Paro, m/z: 109.0,138.0, 329.2,358.2. Solvent delay time was 4 min.
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Table 2
3. Results and Discussion
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3.1. Method Development
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Containing secondary amine groups, Flu, Nor, Map, and Paro can theoretically react with sodium nitrite to form the corresponding N-nitrosamines. Therefore, the chromatographic signals of the original four compounds (20 mg L-1 Flu and Nor, 50
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mg L-1 Map, 100 mg L-1 Paro) and their corresponding derivatives (Flu-NO, Nor-NO, Map-NO, and Paro-NO) were determined by GC-MS in full scan mode and corroborated simultaneously by mass spectrometry. The total ion chromatograms (Fig. 1) show that each substance reacted successfully with nitrite salts under acidic 9
conditions to form derivative products Flu-NO, Nor-NO, Map-NO, and Paro-NO, the chromatographic peak signals after derivatization were significantly enhanced, indicating that the reaction has few by-products, generating a single main stable reaction product, favorable for GC-MS detection. The EI mass spectra of derivative
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products with suggested fragmentation pathways shown in Fig S1. With 2.0 mL of standard solution and fixed sample concentration (50, 50, 125, and 250 μg L-1 for Flu,
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Nor, Map, and Paro, respectively), this study tried to achieve the best results by
optimizing the factors affecting the reaction output, including the derivatization
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acidity, the amount of saturated sodium nitrite solution, the derivatization temperature
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and derivatization time, and the type, volume, and extraction time of the extraction
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solvent.
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Fig. 2
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Fig. 1
The N-nitrosation derivatization reaction requires acidic conditions, so the
hydrochloric acid concentration was first optimized. The concentration effect of 100
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μl HCl on the derivatization reaction was investigated in the range of 0.01 - 0.10 mol L-1. The chromatographic signals of the derivative products Flu-NO, Nor-NO, Map-NO, and Paro-NO increased with the increase of hydrochloric acid concentration within 0.01-0.06 mol L-1 and went down with higher hydrochloric acid concentration 10
(Fig. 2a). Therefore, 100 μl of 0.06 mol L-1 hydrochloric acid was selected as the optimum acidification condition. In the actual sample preparation, due to the weak basicity of plasma and urine, larger amounts of hydrochloric acid were needed (100 μl of 1.00 mol L-1) to achieve the desired recovery yield.
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With other parameters fixed, different volumes (10-200 μL) of saturated sodium nitrite solution was added dropwisely to the acidified working standard solution to
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study the effect of the amount of saturated sodium nitrite solution on the reaction. Maximum derivatization efficiency was achieved at 50 μL of saturated sodium nitrite
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solution (Fig. 2b). Therefore, further experiments were carried out with 50 μL of the
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saturated sodium sulfite solution.
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Derivatization temperature and derivatization time sometimes have a relatively
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large impact on chemical reactions. Therefore, the derivatization efficiency was
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studied under various temperatures: 4 °C, 30 °C, 40 °C, 50 °C, 70 °C, and 80 °C. Fig. 2c shows that the peak area of each derivative first increased and then decreased with
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increasing temperature. Highest efficiency was achieved at 60 °C, which was then
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chosen as the best derivatization temperature. Furthermore, the effect of derivatization time (0-20 min) on the N-nitrosation reaction was investigated at 60 °C (Fig. 2d). The reaction rate was very rapid. When it exceeded 5 min, the reaction time had little
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effect on each derivative product. Because relatively good derivatization efficiency was achieved at 5 min, that time was chosen as the best derivatization time. Choosing the right type and amount of extractant is a critical step in ensuring efficient recovery and high sensitivity. We evaluated the extraction efficiencies of 11
n-hexane, dichloromethane, chloroform, carbon disulfide, ethyl acetate, and toluene under the same volume. Dichloromethane and chloroform gave good extraction efficiencies for Flu-NO. Dichloromethane was also the best extractant for Nor-NO and Map-NO, whereas chloroform was the best for Paro-NO (Fig. 3).
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Dichloromethane was chosen as the extractant. The effect of various dichloromethane volumes (0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 ml) on the extraction efficiency was further
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investigated (Fig. S2). Results showed that the extraction efficiency increased with
increasing volume. However, when the volume of dichloromethane reached 3.0-4.0
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mL, the baseline of the chromatogram increased and the peak areas of the impurities
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increased, causing the sensitivity to decrease. Therefore, 2.0 ml of dichloromethane
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was used to extract all four kinds of derivative products.
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Fig. 3
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3.2 Methodological evaluation
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Under the conditions of optimal chromatographic separation and mass spectrometry, the selective ion chromatograms of the blank and spiked plasma and
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urine samples are shown in Fig. 4. The retention times of Flu-NO, Nor-NO, Map-NO, and Paro-NO were 11.7, 16.2, 18.3, and 23.2 min, respectively, showing great chromatographic separation of the method. It seems that Nor-No has a little overlapping with resulting peaks in the chromatogram of Fig. 4a, by calculating the resolution (R>2.0) and amplifying the chromatogram, it can be concluded that 12
complete separation can be achieved between Nor-No and adjacent peaks. With the selective derivatization method, endogenous substances in the plasma and urine were largely unresponsive and had little interference with the target detection, proving great selectivity of the approach. The result showed that the derived products of Flu, Nor,
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Map, and Paro keep stable within 72 h without obvious decomposition.
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Fig. 4
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Linear calibration curves of Flu, Nor, Map, and Paro were obtained from the
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concentration 0.50-80, 0.50-80, 1.25-200, and 2.5-400 μg L-1, respectively. Very good
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linearity was achieved in the tested calibration ranges with correlation coefficient
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values R2 >0.999 for all the analytes. Method sensitivity was evaluated by
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determining limit of detection (LOD) and the limit of quantitation (LOQ) for the four ATDs. The LOD and LOQ were determined at signal-to-noise (S/N) ratios of 3 and 10,
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respectively. The LODs of Flu, Nor, Map, and Paro were 0.04, 0.08, 0.2, 1.38 μg L-1,
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and LOQs were 0.14, 0.25, 0.67, 4.62 μg L-1, respectively (Table 3).
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Table 3
Table 4
Three concentrations of standard solutions were added to the blank plasma and 13
urine samples (low, medium, and high: 2, 20, and 65 μg L-1 for Flu and Nor, 5, 50, and 162.5 μg L-1 for Map, 10, 100, and 325 μg L-1 for Paro) and tested to verify the precision and accuracy of the method. The data indicated that the intra-day precision (%, RSD) in plasma and urine for Flu, Nor, Map, and Paro at three concentration
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levels were ≤5.28%, and the inter-day were ≤7.87% (Table 4). The results indicated that the method is precise, reliable, and suitable for the detection of Flu, Nor, Map,
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and Paro in plasma and urine. The results showed the method had good precision and accuracy even at the low concentrations. Recovery was measured by spiking known
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amounts of standards in plasma and urine samples in triplicate at three concentration
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levels. Satisfactory values were obtained for both parameters since accuracy ranged
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between 85.88%-118.21% and 80.64%-113.07% in plasma and urine, respectively.
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The occurrence of matrix effects is regarded as a signal suppression or
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enhancement of the analyte due to the coextraction of matrix components, playing an important role in quality of the quantitative data generated by the method. In this
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study, matrix effect was evaluated by comparing calibration curve slope of Flu, Nor,
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Map, and Paro standard solutions in ultra-pure water (A) and matrix-matched standard solutions (B). During method development, the calibration curves were constructed in ultra-pure water, blank plasma and blank urine, respectively. Matrix effect percentage
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is calculated as (B/A) × 100. The results showed that the matrix effect of Flu, Nor, Map, and Paro is 87.75%, 84.41%, 96.20%, and 98.18% in the plasma and 91.87%, 87.38%, 83.06%, and 99.84% in the urine, respectively. Which indicated that there was no significant matrix effect in plasma and urine samples. 14
Table 5 is a comparison of the analytical performance obtained by the proposed method and others for the determination of ATDs in biological samples. Although literature sample separation and enrichment methods can remove the impurities in the matrix for sample purification, there are many shortcomings. For example, disposable extraction
(DPX)extraction
requires
synthesis
polyaniline
and
a
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pipette
styrene-divinylbenzene (SD) copolymer, synthesis takes longer[30]; the operation of
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SPE is complex and tedious, highly reagent consumption and analysis cost[31]; the
fiber of SPME is disposable and expensive[10]; DLLME has many influence factors,
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the most significant factors in DLLME procedure were type and volume of extraction
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and disperser solvents [22, 23, 37]; SDME/MVPE tend to be more technically
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demanding [15]; LC-MS although requires simple protein precipitation(PP) or LLME,
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the instruments are expensive and not widely available in ordinary laboratories[4, 12].
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The previously reported GC analytical methods also require derivatization before analysis, involving butyl chloroformate [20], heptafluorobutyric anhydride, or acetic
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anhydride, which are relatively expensive, require a relatively long derivatization time,
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and are prone to by-products.
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Table 5
The research method reported here overcomes the above disadvantages, plasma and urine samples can be directly derivatized without special pretreatment. Equivalent to a liquid-liquid extraction process, the N-nitrosation derivatization method used in 15
this experiment is inexpensive, rapid, and highly specific. Due to the limitation of sample collection, the target-containing plasma and urine samples from patients were not analyzed in this study. Literatures review showed that the therapeutic concentration of Flu, Nor, Map, and Paro in plasma were 150-500, 50-150, 75-250,
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and 10-75 μg L-1, respectively [35, 36]. The Flu, Map, and Paro intact form is approximately 11%, 3-4%, 2% of intake in urine, Nor is a metabolite of amitriptyline
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[38, 39]. Their urine concentration level reported in the literatures was between tens
to thousands μg L-1 (Table 1) [23, 40-43]. The LOQs of the method (0.14, 0.25, 0.67,
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4.62 μg L-1 for Flu, Nor, Map and Paro, respectively) were significantly lower than
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the plasma and urine actual sample concentrations reported in the literature, which
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demonstrated that the sensitivity of the method meets the requirement for actual
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4. Conclusion
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sample analysis.
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A novel derivatization method was combined with GC-MS to determine the
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contents of Flu, Nor, Map, and Paro in plasma and urine. The derivatization reagent (NaNO2) used is inexpensive, simple, fast, and highly sensitive and specific for secondary amine ATDs. It also shows high accuracy, precision, and without side
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product. This approach can be applied to drug poisoning research, routine clinical and forensic sample analysis, blood drug concentration detection, and assistance for physicians administering medication.
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Conflict of interest The authors have declared no conflict of interest
Acknowledgments
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This work was supported by the Natural Science Foundation of Hebei Province (Nos.
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A
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H 2018206122).
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SC R
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Improvement and validation the method using dispersive liquid-liquid microextraction with in situ derivatization followed by gas chromatography-mass
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SC R
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GC-MS,
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thermal
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ED
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CC E
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[28] I. Papoutsis, A. Khraiwesh, P. Nikolaou, C. Pistos, C. Spiliopoulou, S. Athanaselis, A fully validated method for the simultaneous determination of 11 antidepressant drugs in whole blood by gas chromatography-mass spectrometry, J. Pharmaceut. Biomed. 70 (2012) 557-562. https://doi.org/10.1016/j.jpba.2012.05. 007. 22
[29] D. Ge, H.K. Lee, Ionic liquid based dispersive liquid-liquid microextraction coupled with micro-solid phase extraction of antidepressant drugs from environmental water samples, J. Chromatogr. A 1317 (2013) 217-222. https://doi.org/10.1016/j.chroma.2013.04.014.
Determination
of
Amitriptyline,
Anal.
Chem.
(2015)
8845-8850.
SC R
https://doi.org/10.1021/acs.analchem.5b01895.
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[31] S. Seidi, Y. Yamini, M. Rezazadeh, Combination of electromembrane extraction
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138-146.
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IP T
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SC R
059.
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Int.
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83
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https://doi.org/10.1038/sj.clpt.6100307.
Th.
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analysis,
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Therapeutic drug monitoring of nortriptyline in smoking cessation: a multistudy
M
[37] M.A. Bravo, S. Parra, C. Vargas, W. Quiroz, Determination of organotin
ED
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IP T
https://doi.org/10.1016/j.chroma.2010.10.002. [41] E.E. Chambers, M.J. Woodcock, J.P. Wheatona, T.M. Pekolb, D.M. Diehla,
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Systematic development of an UPLC–MS/MS method for the determination of
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660-665. http://dx.doi.org/10.1016/j.jpba.2013.09.001.
N
[42] M. Prinoth, E. Mtjtschler, Fluorimetric determination of maprotiline in urine and
A
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[43] N. Agrawal, S. M. Peiro, J. E. Romero, A. Durgbanshi, D. Bose, J. P. Vicente, S. C. Broch, Determination of paroxetine in blood and urine using micellar liquid
PT
chromatography with electrochemical detection, J. Chromatogr. Sci. 52 (2014)
A
CC E
1217-1223. https://doi.org/10.1093/chromsci/bmt200.
25
Table 1 Chemical structure, pKa, log P, therapeutic concentration range and urine concentration level of Flu, Nor, Map, and Paro.
log P
Ther. C. (μg/L)
Urine C. (μg/L)
Flu
8.7
4.05
150-500
80-7800
Nor
9.7
4.22
50-150
Map
10.5
4.5
Ref
SC R
[23,35,40]
0.2-346
[23, 41]
36,
75-250
122-913
[35,42]
10-75
840-12340
[35,43]
M
A
N
U
Chemical structure
IP T
pKa
Compound
ED
Paro
9.9
3.95
A
CC E
PT
pKa: dissociation constant, log P: partition coefficient (octanol/water), Ther. C.: therapeutic concentration, Urine C.: urine concentration level.
Table 2 26
The condition of GC programmed temperature. Rate (°C min-1)
Temperature
Maintain
1 2 3 4
10 8 12
130 240 250 260
0 2 0 10
A
CC E
PT
ED
M
A
N
U
SC R
IP T
Step
Table 3
27
Linearity, limit of detection (LOD) and quantification (LOQ) for analysis of Flu, Nor, Map, and Paro. Linear Range (μg L-1)
R2
LOD (μg L-1)
LOQ (μg L-1)
Flu Nor Map Paro
0.50-80 0.50-80 1.25-160 5.0-400
0.9994 0.9995 0.9994 0.9996
0.04 0.08 0.20 1.38
0.14 0.25 0.67 4.62
A
CC E
PT
ED
M
A
N
U
SC R
IP T
Analyte
Table 4 28
Recovery and precision of three spiked concentrations in plasma and urine for Flu,
Analyte
Spiked concentration (μg L−1)
Measured concentration (μg L−1) (n=3)
Recovery (%)
2 20 65 2 20 65 5 50 162.5 10 100 325
2.13±0.02 17.64±0.07 57.35±0.09 2.21±0.24 19.53±0.41 55.92±0.66 4.94±0.80 42.94±2.07 154.13±0.88 11.82±1.20 93.50±2.32 320.28±5.83
105.47 88.20 88.22 110.34 99.58 86.03 98.84 85.88 94.85 118.21 93.50 98.55
2 20 65 2 20 65 5 50 162.5 10 100 325
2.16±0.02 18.83±0.07 60.07±0.09 2.17±0.05 19.55±0.69 57.64±0.25 4.53±0.57 40.32±2.01 134.96±0.96 11.31±1.42 88.91±1.29 342.58±4.20
A
Nor, Map, and Paro. RSD (%) Intra-day (n=6)
Inter-day (n=6)
5.26 3.88 3.22 4.97 3.62 2.03 5.12 4.06 3.00 4.31 5.28 4.71
5.87 6.78 4.28 5.89 7.87 5.74 6.12 7.03 4.92 7.01 6.73 5.74
3.11 4.64 3.63 2.70 1.08 3.72 3.79 1.78 3.68 4.93 3.30 2.00
3.90 6.09 4.84 3.33 1.82 4.48 4.03 2.82 3.80 5.29 4.39 3.78
Paro
A
CC E
Paro
ED
Map
PT
Nor
M
Urine Flu
SC R
Map
U
Nor
N
Flu
Table 5
29
108.08 94.17 92.42 108.73 97.73 88.67 90.69 80.64 83.05 113.07 88.91 105.41
IP T
Plasma
Comparison of the proposed method with previously published methods.
IL-LLME
Dichlorometh ane
Ionic liquids
Derivatization
HCl NaNO2
Samples
and
Plasma and urine
Whole Blood
-
SPME
-
-
Plasma
LC-MS
PP
Acetonitrile
-
CE
SDME/MVPE
Toluene
GC-MS
DLLME
Carbon Tetrachloride
SPE
Acetonitrile
-
Flu
0.50-80
0.14
Nor
0.50-80
0.25
Map
1.25-200
0.67
Paro
2.5-400
4.62
Flu
10-1000
10
Nor
10-1000
Map
10-1000
Paro
10-1000
Flu
25-1200
10 10
Reference
This study
[4]
10 25
[10]
16-1200
16
Human milk
Flu
5-320
5.0
Paro
20-600
20
-
Urine
Nor
2.5-62.5
2.5
[15]
Acetic Anhydride
Urine
Nor
2-100
0.50
[22]
Flu
8-100000
7.0
Nor
8-100000
5.0
Flu
30-10000
27
Nor
20-10000
18
Flu
10-1000
10
Flu
5-1000
1.0
Nor
5-1000
1.0
Map
5-1000
1.0
Paro
5-1000
5.0
N
Urine Plasma
M
Butyl Chloroformate
-
Heptafluorobut yric Anhydride
Plasma
Whole blood
[12]
[23]
[27]
[28]
CC E
GC-MS
DPX
1,1,2,2-tetrach loroethane
LOQ (μg L-1)
Paro
ED
LC-FD
DLLME
PT
GC-FID
Linear range (μg L-1)
A
LC-UV
Analytes
IP T
LC-MS/M S
-
Extraction Solvent
SC R
GC-MS
Extraction Method
U
Instrumen t
GC-MS: gas chromatography- mass spectrometry, LC-MS/MS: liquid chromatography-tandem mass spectrometry, LC-UV: liquid chromatography-ultraviolet detector, LC-MS: liquid chromatography-mass spectrometry, CE: capillary electrophoresis, LC-FD: liquid chromatography-fluorescence detector, GC-FID: gas chromatography-flame ionization detection.IL-LLME: Ionic
A
Liquid-Liquid-Liquid Microextraction, SPME: Solid-Phase Microextraction, PP: Protein Precipitation, SDME/MVPE: Single-Drop Microextraction/Micro-Volume Pipette Extraction, DPX: Disposable Pipette Extraction, SPE: Solid-Phase Extraction, DLLME: Dispersive Liquid-Liquid Microextraction.
30
Figure Captions Fig. 1. The total ion current chromatogram of the Flu, Nor, Map, and Paro and their respective derivatives derivative. Fig. 2. The effects of hydrochloric acid concentration (a), the amount of saturated
IP T
NaNO2 (b), derivatization temperature (c), and derivatization time (d) on the peak
Fig. 3. The extraction efficiency of six different solvents.
SC R
area of the four derivatives.
Fig. 4. The selected-ion monitoring mode chromatograms of blank plasma samples
U
and spiked plasma samples (a) and blank urine samples and spiked urine samples (b).
A
CC E
PT
ED
M
A
N
(20 μg L-1 for Flu and Nor, 50 μg L-1 for Map, 100 μg L-1 for Paro).
31
Abundance
Flu-NO
2400000
Nor-NO Map-NO
2000000
Paro-NO
800000
Flu Nor
Map
SC R
1200000
Paro
10.00
14.00
U
400000 6.00
18.00
22.00
N
Time
A
CC E
PT
ED
M
A
Figure 1
32
IP T
1600000
26.00
7x10
5
4x10
5
3x10
5
0.02
0.04
0.06
0.08
6
1x10
6
9x10
5
8x10
5
4x10
5
0.10
0
1.4x10
6
1.2x10
6
1.0x10
6
6.0x10
5
6
1.05x10
6
9.00x10
5
5x10
5
4x10
5
5
0
10
20
30
40
50
60
70
80
90
0
N
4.0x10
1.20x10 P e a k a re a
P e a k a re a
6
A
D erivatization tem perature C
A
CC E
PT
ED
M
Figure 2
100
150
200
A m o u n t o f satu rated N aN O 2 ( L )
Concentration of 100L HCl (mol/L) 1.6x10
50
IP T
8x10
5
1x10
SC R
9x10
5
U
6
P e a k a re a
Peak Area
1x10
33
5
10
15
Derivatization tim e m in
20
1.2x10
6
9.0x10
5
6.0x10
5
3.0x10
5
Flu Nor Map Paro
IP T
6
SC R
Peak aera
1.5x10
0.0
U
e e e ne ene tat orm lfid xen u ha e f l t e u c o o e s i T la m lor n-H nd thy o oro Ch l b E h r Ca Dic
A
CC E
PT
ED
M
A
N
Figure 3
34
(a) Blank and Spiked Plasma Flu-NO
Nor-NO 18000
Abundance 30000
2000 16.50
IP T
16.00
24000
Map-NO
18000
Paro-NO
6000 2000 12.00
14.00
16.00
18.00
20.00
22.00
24.00
U
10.00 Time -->
SC R
12000
N
(b) Blank and Spiked Urine Flu-NO 18000
A
Nor-NO
30000 2000
24000
M
Abundance
16.50
ED
16.00
18000
6000
CC E
2000
PT
12000
Paro-NO
Map-NO
12.00
14.00
16.00
A
10.00 Time -->
Figure 4
35
18.00
20.00
22.00
24.00
S0021-9673(19)30406-6 https://doi.org/10.1016/j.chroma.2019.04.038 CHROMA 360184
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
10 January 2019 11 April 2019 13 April 2019
Please cite this article as: Feng Y, Zheng M, Zhang X, Kang K, Kang W, Yang J, Lian K, Analysis of Four Antidepressants in Plasma and Urine by Gas ChromatographyMass Spectrometry Combined with Sensitive and Selective Derivatization, Journal of Chromatography A (2019), https://doi.org/10.1016/j.chroma.2019.04.038 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Analysis of Four Antidepressants in Plasma and Urine by Gas Chromatography-Mass Spectrometry Combined with Sensitive and Selective Derivatization
c,
**,
IP T
Yanru Feng a, Min Zheng a, Xue Zhang a, Kai Kang b, Weijun Kang a, Jie Yang
SC R
Kaoqi Liana, d, *
School of Public Health, Hebei Medical University, Shijiazhuang, 050017, China
b
School of Pharmacy, Hebei Medical University, Shijiazhuang, 050017, China
c
The pharmaceutical department of the Third Hospital of Hebei Medical University,
N
U
a
Hebei Key Laboratory of Environment and Human Health, Shijiazhuang 050017,
M
d
A
Shijiazhuang 050051, China
ED
China
PT
*Corresponding author:
CC E
Department of Sanitary Inspection, School of Public Health, Hebei Medical University, Shijiazhuang, P. R. China. E-mail addresses: [email protected] (K.Q. Lian)
A
** Corresponding author: The pharmaceutical department of the Third Hospital of Hebei Medical University Shijiazhuang, P. R. China.E-mail addresses: [email protected] (J. Yang)
1
Highlights
A simple and fast derivatization method was used to analysis of four ATDs by GC-MS.
The method also provided excellent sensitivity and selective without side
IP T
product. The LOQs of the analytes were significantly lower than actual sample
SC R
concentration.
The method will provide a new strategy for the detection of ATDs in plasma
N
U
and urine.
A
ABSTRACT
M
A sensitive and selective method was developed for simultaneous determination
ED
of four antidepressants (ATDs) in plasma and urine samples by gas chromatographymass spectrometry (GC-MS) based on an N-nitrosation reaction. In this study,
PT
fluoxetine (Flu), nortriptyline (Nor), maprotiline (Map), and paroxetine (Paro) were
CC E
first derivatized with sodium nitrite to appropriate N-nitrosamines under acidic condition, then the derivatives were easier to detect by GC-MS. The derivatization conditions including the amount of hydrochloric acid, the amount of sodium nitrite,
A
reaction temperature, reaction time and the extraction reagents were optimized. Under the optimal conditions, the limit of detections (LODs) and limit of quantitations (LOQs) were in the range of 0.04-1.38 μg L-1 and 0.14-4.62 μg L-1, respectively. Low, medium, and high concentrations of antidepressants were added in plasma and urine 2
samples, spiked recovery ranged from 85.88% to 110.34% for plasma and 80.64% to 113.07% for urine, respectively. The derivatization reaction was very quickly, only 5 min was needed for the reaction process, in addition, the proposed method exhibited superior sensitivity and selectivity, it showed sufficient advantages for determination
IP T
of Flu, Nor, Map, and Paro in plasma and urine of patients.
SC R
Keywords: antidepressants, derivatization, Gas chromatography-mass spectrometry,
U
N-Nitrosamine, plasma, urine
N
1. Introduction
A
Depression is one of the most common chronic or recurrent mental illnesses in
M
modern society. It is often characterized by inattention, reduced self-confidence,
ED
pessimism, guilt, sleep disturbance, self-harm, and ultimately suicide [1, 2]. antidepressants (ATDs) are widely used to alleviate and treat symptoms of mental
PT
illness. Due to their side effects, toxicity and severe drug interactions [3],
CC E
first-generation antidepressants such as tricyclic antidepressants (TCAs) are gradually being replaced by a new generation of ATDs, such as selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) [4].
A
However, due to the narrow safety window of antidepressant drugs and the large patient variation, patients taking medication need long-term monitoring of vivo concentration [5, 6]. At the same time, due to the poor compliance of patients with depression, patients often suffer from poisoning due to overdose or misuse [7]. 3
Therefore, the establishment of sensitive, rapid, and reliable analytical detection techniques for ATDs is of great significance for identifying drug poisoning, monitoring therapeutical drugs, assisting clinicians to adjust drug dosage, and achieving individualized rational use of medications [8, 9]. performance
liquid
chromatography
(HPLC)
[10,
11],
liquid
IP T
High
chromatography-mass spectrometry (LC-MS) [12-14], and capillary electrophoresis
SC R
(CE) [15, 16] have been used for the analysis and detection of ATDs. The biological samples involved are mainly plasma, urine and whole blood. Due to the complex
U
interference of the biological sample matrix, separation and enrichment techniques are
N
needed for processing. The previously reported sample pretreatment methods are
phase
micro-extraction
(SPME)
M
solid
A
liquid-liquid extraction (LLE) [17, 18], solid phase extraction (SPE) [16, 19, 20], [10,
21],
dispersive
liquid-liquid
ED
microextraction (DLLME) [22, 23]; liquid phase microextraction (LPME) [24], ionic liquid-liquid-liquid microextraction (IL-LLME) [4]; stir bar sorptive extraction (SBSE)
PT
[25, 26], single-drop microextraction/microvolume pipette extraction (SDME/MVPE)
CC E
[15], and the others [4, 27-31], which have the disadvantages of troublesome operation, lengthy procedure , high cost, disposable, and high consumption of organic
A
solvents.
Gas chromatography (GC) is a commonly used method in mixture analysis. It
has the characteristics and advantages of mature technology, easy to master, high detection sensitivity, high separation efficiency, high selectivity, low detection limit, low sample consumption, rapidness, and convenience. However, ideal compounds for 4
GC analysis should have low boiling points, good thermal stability, and low molecular polarities. In order to convert compounds that are either undetectable directly by GC or detected with a low sensitivity to those ideal ones that are easily detected by GC analysis, chemical derivatization techniques are commonly used, including acylation
IP T
[22, 32], silanization [33], and esterification [23]. Acylation is the conversion of a compound containing active hydrogen into an ester, sulfide, or amide by means of a
SC R
carboxylic acid; silanization is generally the replacement of active hydrogen by
silylation; esterification is the reaction of alcohols with carboxylic acids to form esters.
U
There are several reports on the application of GC in combination with derivatization
N
technology to ATDs. However, these derivatization methods have the problems of
A
relatively complicated operation procedures, long reaction times, and numerous side
M
reactions.
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The authors previously established an innovative new method for the detection of ketamine in urine and plasma with gas chromatography-mass spectrometry
PT
(GC-MS) by using the reaction of secondary amines and sodium nitrite to form
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N-nitrosamine compounds under acidic conditions [34]. This derivatization method has three major advantages. First, the product N-nitrosamines are fat-soluble substances, making them readily extracted by organic solvents; being less polar, the
A
products are very suitable for GC-MS detection with greatly improved detection sensitivity. Second, since only secondary amine molecules can be quantitatively reacted in this reaction, a large amount of matrix interference can be eliminated, greatly improving selectivity. Last, this reaction is fast, shortening the analysis cycle. 5
Table 1
Many ATDs such as fluoxetine (Flu), nortriptyline (Nor), maprotiline (Map), and
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paroxetine (Paro) were found to contain secondary amine groups, the chemical structure, some physicochemical properties (pKa, log P) of Flu, Nor, Map, and Paro
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were shown in Table 1 [35, 36]. Previous studies have found that the sensitivity of direct extraction from biological sample followed by GC-MS detection was very low.
U
Theoretically, such ATDs with secondary amines can react with sodium nitrite under
N
acidic conditions to form corresponding N-nitrosamine. However, application of this
A
derivatization approach with gas chromatography or gas chromatography-mass
M
spectrometry analysis of ATDs has not been reported. Through optimization of the
ED
reaction conditions, extraction parameters, and GC-MS separation and analysis procedures, we have established a high-sensitivity and high-selectivity GC-MS
PT
approach for the detection of Flu, Nor, Map, and Paro in plasma and urine. This
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method may be applied to the detection of similar clinical drug content and side effect
A
control, and play a positive role in assisting clinicians to adjust the drug dosage.
2. Experimental 2.1. Chemicals and reagents Nortriptyline hydrochloride (Nor) was purchased from Sigma (St. Louis, MO, USA), Fluoxetine hydrochloride (Flu), Maprotiline hydrochloride (Map) and 6
Paroxetine hydrochloride (Paro) were bought from National Institutes for Food and Drug control (Beijing, China). Analytical reagent grade sodium nitrite (NaNO2) and hydrochloric acid (HCl) were from Hubei university chemical factory (Hubei, China) or Shijiazhuang Huadi chemical industry and trade co. LTD. (Shijiazhuang, China)
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were used as derivatizing reagent. Analytical grade chloroform, dichloromethane, carbon disulfide obtained from Xilong Chemical Factory (Shantou, China) or Tianjin
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General Chemical Reagent Factory (Tianjin, China) and HPLC grade n-hexane, ethyl acetate, toluene supplied by Tianjin commie chemical reagent co. LTD. (Tianjin,
U
China) or Li’anlongbohua pharmaceutical chemical co. LTD (Tianjin, China). were
N
tested as extraction solvents. Methanol (MeOH, HPLC grade) was supplied by
A
Oceanpakalexative chemical., Ltd (Gothenburg, Sweden). Ultra-pure water was
M
obtained from Sichuan Youpu super pure technology co. LTD. UPR-II-20L (Sichuan,
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China).
2.2. Preparation of standard and working solutions
PT
Primary stock solutions of each individual drug (Flu, Nor, Map, and Paro) were
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prepared in MeOH at a concentration of 1 mg ml-1 and stored light protected at -20℃. Standards working solutions of the mixtures of each compound at 50, 50, 125 and 250 μg L-1 (Flu, Nor, Map, and Paro) respectively were prepared by proper dilutions with
A
ultrapure water.
2.3. Sample Preparation Human blank plasma and urine were stored at -20 °C for the preparation of quality control (QC) samples. Three concentrations of QC samples were prepared in 7
plasma and urine (low, medium, and high): 2, 20, and 65 μg L-1 for Flu and Nor, 5, 50, and 162.5 μg L-1 for Map, and 10, 100, and 325 μg L-1 for Paro, respectively. The sample solutions were filtered through a 0.45 μm filter before derivatization. 2.4. Derivatization
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Into a 10.0 mL open glass tube, 2.0 mL of standard solution or sample solution was added followed by the addition of 100 μL of 1.0 mol L-1 HCl, vortexed prior to
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the addition of 50 μL of saturated sodium nitrite solution dropwise under vortexing.
The sample tube was then sealed and heated at 60 °C for 5 min to carry out the
U
derivatization reaction. After cooling, 2.0 mL of dichloromethane was added for
N
extraction. The mixture was vortexed for 1.5 min, allowed to stand for 5 min for
A
stratification (plasma and urine samples were centrifuged at 15000 rpm for 5 min).
M
With a micropipette, 1.2 mL of the organic phase was transferred into a glass tube
ED
with a stopper, and blow-dried with nitrogen at 50 °C. The residue was dissolved in 50 μL of dichloromethane and placed in an ice bath to prevent dichloromethane
PT
volatilization, 1 μL of which was taken for GC-MS analysis.
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2.5. GC-MS analysis
GC-MS analysis was performed on an Agilent (Little Falls, DE, USA) gas
chromatograph 7890A equipped with an electronically controlled split/splitless
A
injection port, an inert 5975C mass selective detector with electron impact (EI) ionization chamber, and a 7683B Series injector/autosampler. Chromatographic separation was conducted with an HP-5 MS 30 m×0.25 mm I.D., 0.25 μm film thickness column (Agilent, CA, USA).GC oven programmed 8
temperature condition was shown in Table 2.Injections (1μL) were done at 250 °C in the split mode, split ratio was 5:1. Helium was used as the carrier gas with constant flow rate at 1.2 mL min-1. The mass spectrometer (MS) was operated at electronic impact (EI, 70 eV) mode, ion source temperature at 230 °C, and MS quadrupole
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temperature at 150 °C. The MS transfer line temperature was held at 280 °C. In the full-scan mode, preliminary mass spectra of the target analytes were obtained from
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m/z 0 to 750 AMU. Selected-ion monitoring mode used for the selectivity and sensitivity concerns, the monitored ions were selected as follows: Flu, m/z: 104.1,
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117.1, 177.1; Nor, m/z: 204.1, 219.1, 232.1; Map, m/z: 178.1, 203.1,248.2, 276.1;
A
N
Paro, m/z: 109.0,138.0, 329.2,358.2. Solvent delay time was 4 min.
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M
Table 2
3. Results and Discussion
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3.1. Method Development
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Containing secondary amine groups, Flu, Nor, Map, and Paro can theoretically react with sodium nitrite to form the corresponding N-nitrosamines. Therefore, the chromatographic signals of the original four compounds (20 mg L-1 Flu and Nor, 50
A
mg L-1 Map, 100 mg L-1 Paro) and their corresponding derivatives (Flu-NO, Nor-NO, Map-NO, and Paro-NO) were determined by GC-MS in full scan mode and corroborated simultaneously by mass spectrometry. The total ion chromatograms (Fig. 1) show that each substance reacted successfully with nitrite salts under acidic 9
conditions to form derivative products Flu-NO, Nor-NO, Map-NO, and Paro-NO, the chromatographic peak signals after derivatization were significantly enhanced, indicating that the reaction has few by-products, generating a single main stable reaction product, favorable for GC-MS detection. The EI mass spectra of derivative
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products with suggested fragmentation pathways shown in Fig S1. With 2.0 mL of standard solution and fixed sample concentration (50, 50, 125, and 250 μg L-1 for Flu,
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Nor, Map, and Paro, respectively), this study tried to achieve the best results by
optimizing the factors affecting the reaction output, including the derivatization
U
acidity, the amount of saturated sodium nitrite solution, the derivatization temperature
N
and derivatization time, and the type, volume, and extraction time of the extraction
M
A
solvent.
PT
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Fig. 2
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Fig. 1
The N-nitrosation derivatization reaction requires acidic conditions, so the
hydrochloric acid concentration was first optimized. The concentration effect of 100
A
μl HCl on the derivatization reaction was investigated in the range of 0.01 - 0.10 mol L-1. The chromatographic signals of the derivative products Flu-NO, Nor-NO, Map-NO, and Paro-NO increased with the increase of hydrochloric acid concentration within 0.01-0.06 mol L-1 and went down with higher hydrochloric acid concentration 10
(Fig. 2a). Therefore, 100 μl of 0.06 mol L-1 hydrochloric acid was selected as the optimum acidification condition. In the actual sample preparation, due to the weak basicity of plasma and urine, larger amounts of hydrochloric acid were needed (100 μl of 1.00 mol L-1) to achieve the desired recovery yield.
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With other parameters fixed, different volumes (10-200 μL) of saturated sodium nitrite solution was added dropwisely to the acidified working standard solution to
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study the effect of the amount of saturated sodium nitrite solution on the reaction. Maximum derivatization efficiency was achieved at 50 μL of saturated sodium nitrite
U
solution (Fig. 2b). Therefore, further experiments were carried out with 50 μL of the
N
saturated sodium sulfite solution.
A
Derivatization temperature and derivatization time sometimes have a relatively
M
large impact on chemical reactions. Therefore, the derivatization efficiency was
ED
studied under various temperatures: 4 °C, 30 °C, 40 °C, 50 °C, 70 °C, and 80 °C. Fig. 2c shows that the peak area of each derivative first increased and then decreased with
PT
increasing temperature. Highest efficiency was achieved at 60 °C, which was then
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chosen as the best derivatization temperature. Furthermore, the effect of derivatization time (0-20 min) on the N-nitrosation reaction was investigated at 60 °C (Fig. 2d). The reaction rate was very rapid. When it exceeded 5 min, the reaction time had little
A
effect on each derivative product. Because relatively good derivatization efficiency was achieved at 5 min, that time was chosen as the best derivatization time. Choosing the right type and amount of extractant is a critical step in ensuring efficient recovery and high sensitivity. We evaluated the extraction efficiencies of 11
n-hexane, dichloromethane, chloroform, carbon disulfide, ethyl acetate, and toluene under the same volume. Dichloromethane and chloroform gave good extraction efficiencies for Flu-NO. Dichloromethane was also the best extractant for Nor-NO and Map-NO, whereas chloroform was the best for Paro-NO (Fig. 3).
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Dichloromethane was chosen as the extractant. The effect of various dichloromethane volumes (0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 ml) on the extraction efficiency was further
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investigated (Fig. S2). Results showed that the extraction efficiency increased with
increasing volume. However, when the volume of dichloromethane reached 3.0-4.0
U
mL, the baseline of the chromatogram increased and the peak areas of the impurities
N
increased, causing the sensitivity to decrease. Therefore, 2.0 ml of dichloromethane
M
A
was used to extract all four kinds of derivative products.
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Fig. 3
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3.2 Methodological evaluation
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Under the conditions of optimal chromatographic separation and mass spectrometry, the selective ion chromatograms of the blank and spiked plasma and
A
urine samples are shown in Fig. 4. The retention times of Flu-NO, Nor-NO, Map-NO, and Paro-NO were 11.7, 16.2, 18.3, and 23.2 min, respectively, showing great chromatographic separation of the method. It seems that Nor-No has a little overlapping with resulting peaks in the chromatogram of Fig. 4a, by calculating the resolution (R>2.0) and amplifying the chromatogram, it can be concluded that 12
complete separation can be achieved between Nor-No and adjacent peaks. With the selective derivatization method, endogenous substances in the plasma and urine were largely unresponsive and had little interference with the target detection, proving great selectivity of the approach. The result showed that the derived products of Flu, Nor,
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Map, and Paro keep stable within 72 h without obvious decomposition.
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Fig. 4
U
Linear calibration curves of Flu, Nor, Map, and Paro were obtained from the
N
concentration 0.50-80, 0.50-80, 1.25-200, and 2.5-400 μg L-1, respectively. Very good
A
linearity was achieved in the tested calibration ranges with correlation coefficient
M
values R2 >0.999 for all the analytes. Method sensitivity was evaluated by
ED
determining limit of detection (LOD) and the limit of quantitation (LOQ) for the four ATDs. The LOD and LOQ were determined at signal-to-noise (S/N) ratios of 3 and 10,
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respectively. The LODs of Flu, Nor, Map, and Paro were 0.04, 0.08, 0.2, 1.38 μg L-1,
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and LOQs were 0.14, 0.25, 0.67, 4.62 μg L-1, respectively (Table 3).
A
Table 3
Table 4
Three concentrations of standard solutions were added to the blank plasma and 13
urine samples (low, medium, and high: 2, 20, and 65 μg L-1 for Flu and Nor, 5, 50, and 162.5 μg L-1 for Map, 10, 100, and 325 μg L-1 for Paro) and tested to verify the precision and accuracy of the method. The data indicated that the intra-day precision (%, RSD) in plasma and urine for Flu, Nor, Map, and Paro at three concentration
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levels were ≤5.28%, and the inter-day were ≤7.87% (Table 4). The results indicated that the method is precise, reliable, and suitable for the detection of Flu, Nor, Map,
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and Paro in plasma and urine. The results showed the method had good precision and accuracy even at the low concentrations. Recovery was measured by spiking known
U
amounts of standards in plasma and urine samples in triplicate at three concentration
N
levels. Satisfactory values were obtained for both parameters since accuracy ranged
A
between 85.88%-118.21% and 80.64%-113.07% in plasma and urine, respectively.
M
The occurrence of matrix effects is regarded as a signal suppression or
ED
enhancement of the analyte due to the coextraction of matrix components, playing an important role in quality of the quantitative data generated by the method. In this
PT
study, matrix effect was evaluated by comparing calibration curve slope of Flu, Nor,
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Map, and Paro standard solutions in ultra-pure water (A) and matrix-matched standard solutions (B). During method development, the calibration curves were constructed in ultra-pure water, blank plasma and blank urine, respectively. Matrix effect percentage
A
is calculated as (B/A) × 100. The results showed that the matrix effect of Flu, Nor, Map, and Paro is 87.75%, 84.41%, 96.20%, and 98.18% in the plasma and 91.87%, 87.38%, 83.06%, and 99.84% in the urine, respectively. Which indicated that there was no significant matrix effect in plasma and urine samples. 14
Table 5 is a comparison of the analytical performance obtained by the proposed method and others for the determination of ATDs in biological samples. Although literature sample separation and enrichment methods can remove the impurities in the matrix for sample purification, there are many shortcomings. For example, disposable extraction
(DPX)extraction
requires
synthesis
polyaniline
and
a
IP T
pipette
styrene-divinylbenzene (SD) copolymer, synthesis takes longer[30]; the operation of
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SPE is complex and tedious, highly reagent consumption and analysis cost[31]; the
fiber of SPME is disposable and expensive[10]; DLLME has many influence factors,
U
the most significant factors in DLLME procedure were type and volume of extraction
N
and disperser solvents [22, 23, 37]; SDME/MVPE tend to be more technically
A
demanding [15]; LC-MS although requires simple protein precipitation(PP) or LLME,
M
the instruments are expensive and not widely available in ordinary laboratories[4, 12].
ED
The previously reported GC analytical methods also require derivatization before analysis, involving butyl chloroformate [20], heptafluorobutyric anhydride, or acetic
PT
anhydride, which are relatively expensive, require a relatively long derivatization time,
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and are prone to by-products.
A
Table 5
The research method reported here overcomes the above disadvantages, plasma and urine samples can be directly derivatized without special pretreatment. Equivalent to a liquid-liquid extraction process, the N-nitrosation derivatization method used in 15
this experiment is inexpensive, rapid, and highly specific. Due to the limitation of sample collection, the target-containing plasma and urine samples from patients were not analyzed in this study. Literatures review showed that the therapeutic concentration of Flu, Nor, Map, and Paro in plasma were 150-500, 50-150, 75-250,
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and 10-75 μg L-1, respectively [35, 36]. The Flu, Map, and Paro intact form is approximately 11%, 3-4%, 2% of intake in urine, Nor is a metabolite of amitriptyline
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[38, 39]. Their urine concentration level reported in the literatures was between tens
to thousands μg L-1 (Table 1) [23, 40-43]. The LOQs of the method (0.14, 0.25, 0.67,
U
4.62 μg L-1 for Flu, Nor, Map and Paro, respectively) were significantly lower than
N
the plasma and urine actual sample concentrations reported in the literature, which
A
demonstrated that the sensitivity of the method meets the requirement for actual
ED
4. Conclusion
M
sample analysis.
PT
A novel derivatization method was combined with GC-MS to determine the
CC E
contents of Flu, Nor, Map, and Paro in plasma and urine. The derivatization reagent (NaNO2) used is inexpensive, simple, fast, and highly sensitive and specific for secondary amine ATDs. It also shows high accuracy, precision, and without side
A
product. This approach can be applied to drug poisoning research, routine clinical and forensic sample analysis, blood drug concentration detection, and assistance for physicians administering medication.
16
Conflict of interest The authors have declared no conflict of interest
Acknowledgments
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This work was supported by the Natural Science Foundation of Hebei Province (Nos.
A
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PT
ED
M
A
N
U
SC R
H 2018206122).
17
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CC E
5683-5687. https://doi.org/10.1016/j.chroma.2011.06.022. [33] S. Casal, E. Mendes, J.O. Fernandes, M.B.P.P. Oliveira, M.A. Ferreira, Analysis
A
of heterocyclic aromatic amines in foods by gas chromatography–mass spectrometry as their tert.-butyldimethylsilyl derivatives,J. Chromatogr. A 1040 (2004) 105-114. https://doi.org/10.1016/j.chroma.2004.03.054.01.004.
[34] K. Lian, P. Zhang, L. Niu, S. Bi, S. Liu, L. Jiang, W. Kang, A novel derivatization approach for determination of ketamine in urine and plasma by gas 23
chromatography-mass spectrometry, J. Chromatogr. A 1264 (2012) 104-109. https://doi.org/10.1016/j.chroma.2012.09.058. [35] S.M.R. Wille, K.E. Maudens, C.H. Van Peteghem, W.E.E. Lambert, Development of a solid phase extraction for 13 new generation antidepressants
IP T
and their active metabolites for gas chromatographic-mass spectrometric analysis, J. Chromatogr. A 1098 (2005) 19-29. https://doi.org/10.1016/j.chroma.2005.08.
SC R
059.
[36] M.E. Mooney, V.I. Reus, J. Gorecki, S.M. Hall, G.L. Humfleet, R.F. Muñoz, etc,
Int.
J.
Clin.
Pharm.
83
(2008)
436-442.
A
https://doi.org/10.1038/sj.clpt.6100307.
Th.
N
analysis,
U
Therapeutic drug monitoring of nortriptyline in smoking cessation: a multistudy
M
[37] M.A. Bravo, S. Parra, C. Vargas, W. Quiroz, Determination of organotin
ED
compounds in sediment samples by dispersive liquid-liquid microextraction followed by gas chromatography-pulsed flame photometric detection, Microchem.
PT
J. 134 (2017) 49-53. https://doi.org/10.1016/j.microc.2017.05.009.
CC E
[38] H. Bagheri, O. Zandi, A. Aghakhani, Extraction of fluoxetine from aquatic and urine samples using sodium dodecyl sulfate-coated iron oxide magnetic
A
nanoparticles followed by spectrofluorimetric determination, 716 (2012) 61-65. https://doi.org/10.1016/j.aca.2011.10.033.
[39] E.H. William, Paroxetine: a review of its pharmacology, pharmacokinetics and utility in the treatment of a variety of psychiatric disorders, 8 (1999) 417-441. https://doi.org/10.1517/13543784.8.4.417. 24
[40] M.M. Zheng, S.T. Wang, W.K. Hu, Y.Q. Feng, In-tube solid-phase microextraction
based
on
hybrid
silica
monolith
coupled
to
liquid
chromatography–mass spectrometry for automated analysis of ten antidepressants in human urine and plasma, J. Chromatogr. A 1217 (2010) 7493-7501.
IP T
https://doi.org/10.1016/j.chroma.2010.10.002. [41] E.E. Chambers, M.J. Woodcock, J.P. Wheatona, T.M. Pekolb, D.M. Diehla,
SC R
Systematic development of an UPLC–MS/MS method for the determination of
tricyclic antidepressants in human urine, J. Pharmaceut. Biomed. 88 (2014)
U
660-665. http://dx.doi.org/10.1016/j.jpba.2013.09.001.
N
[42] M. Prinoth, E. Mtjtschler, Fluorimetric determination of maprotiline in urine and
A
plasma after thin-layer chromatographic separation, J. Chromatogr. B 305 (1984)
M
508-511. https://doi.org/10.1016/S0378-4347(00)83370-8.
ED
[43] N. Agrawal, S. M. Peiro, J. E. Romero, A. Durgbanshi, D. Bose, J. P. Vicente, S. C. Broch, Determination of paroxetine in blood and urine using micellar liquid
PT
chromatography with electrochemical detection, J. Chromatogr. Sci. 52 (2014)
A
CC E
1217-1223. https://doi.org/10.1093/chromsci/bmt200.
25
Table 1 Chemical structure, pKa, log P, therapeutic concentration range and urine concentration level of Flu, Nor, Map, and Paro.
log P
Ther. C. (μg/L)
Urine C. (μg/L)
Flu
8.7
4.05
150-500
80-7800
Nor
9.7
4.22
50-150
Map
10.5
4.5
Ref
SC R
[23,35,40]
0.2-346
[23, 41]
36,
75-250
122-913
[35,42]
10-75
840-12340
[35,43]
M
A
N
U
Chemical structure
IP T
pKa
Compound
ED
Paro
9.9
3.95
A
CC E
PT
pKa: dissociation constant, log P: partition coefficient (octanol/water), Ther. C.: therapeutic concentration, Urine C.: urine concentration level.
Table 2 26
The condition of GC programmed temperature. Rate (°C min-1)
Temperature
Maintain
1 2 3 4
10 8 12
130 240 250 260
0 2 0 10
A
CC E
PT
ED
M
A
N
U
SC R
IP T
Step
Table 3
27
Linearity, limit of detection (LOD) and quantification (LOQ) for analysis of Flu, Nor, Map, and Paro. Linear Range (μg L-1)
R2
LOD (μg L-1)
LOQ (μg L-1)
Flu Nor Map Paro
0.50-80 0.50-80 1.25-160 5.0-400
0.9994 0.9995 0.9994 0.9996
0.04 0.08 0.20 1.38
0.14 0.25 0.67 4.62
A
CC E
PT
ED
M
A
N
U
SC R
IP T
Analyte
Table 4 28
Recovery and precision of three spiked concentrations in plasma and urine for Flu,
Analyte
Spiked concentration (μg L−1)
Measured concentration (μg L−1) (n=3)
Recovery (%)
2 20 65 2 20 65 5 50 162.5 10 100 325
2.13±0.02 17.64±0.07 57.35±0.09 2.21±0.24 19.53±0.41 55.92±0.66 4.94±0.80 42.94±2.07 154.13±0.88 11.82±1.20 93.50±2.32 320.28±5.83
105.47 88.20 88.22 110.34 99.58 86.03 98.84 85.88 94.85 118.21 93.50 98.55
2 20 65 2 20 65 5 50 162.5 10 100 325
2.16±0.02 18.83±0.07 60.07±0.09 2.17±0.05 19.55±0.69 57.64±0.25 4.53±0.57 40.32±2.01 134.96±0.96 11.31±1.42 88.91±1.29 342.58±4.20
A
Nor, Map, and Paro. RSD (%) Intra-day (n=6)
Inter-day (n=6)
5.26 3.88 3.22 4.97 3.62 2.03 5.12 4.06 3.00 4.31 5.28 4.71
5.87 6.78 4.28 5.89 7.87 5.74 6.12 7.03 4.92 7.01 6.73 5.74
3.11 4.64 3.63 2.70 1.08 3.72 3.79 1.78 3.68 4.93 3.30 2.00
3.90 6.09 4.84 3.33 1.82 4.48 4.03 2.82 3.80 5.29 4.39 3.78
Paro
A
CC E
Paro
ED
Map
PT
Nor
M
Urine Flu
SC R
Map
U
Nor
N
Flu
Table 5
29
108.08 94.17 92.42 108.73 97.73 88.67 90.69 80.64 83.05 113.07 88.91 105.41
IP T
Plasma
Comparison of the proposed method with previously published methods.
IL-LLME
Dichlorometh ane
Ionic liquids
Derivatization
HCl NaNO2
Samples
and
Plasma and urine
Whole Blood
-
SPME
-
-
Plasma
LC-MS
PP
Acetonitrile
-
CE
SDME/MVPE
Toluene
GC-MS
DLLME
Carbon Tetrachloride
SPE
Acetonitrile
-
Flu
0.50-80
0.14
Nor
0.50-80
0.25
Map
1.25-200
0.67
Paro
2.5-400
4.62
Flu
10-1000
10
Nor
10-1000
Map
10-1000
Paro
10-1000
Flu
25-1200
10 10
Reference
This study
[4]
10 25
[10]
16-1200
16
Human milk
Flu
5-320
5.0
Paro
20-600
20
-
Urine
Nor
2.5-62.5
2.5
[15]
Acetic Anhydride
Urine
Nor
2-100
0.50
[22]
Flu
8-100000
7.0
Nor
8-100000
5.0
Flu
30-10000
27
Nor
20-10000
18
Flu
10-1000
10
Flu
5-1000
1.0
Nor
5-1000
1.0
Map
5-1000
1.0
Paro
5-1000
5.0
N
Urine Plasma
M
Butyl Chloroformate
-
Heptafluorobut yric Anhydride
Plasma
Whole blood
[12]
[23]
[27]
[28]
CC E
GC-MS
DPX
1,1,2,2-tetrach loroethane
LOQ (μg L-1)
Paro
ED
LC-FD
DLLME
PT
GC-FID
Linear range (μg L-1)
A
LC-UV
Analytes
IP T
LC-MS/M S
-
Extraction Solvent
SC R
GC-MS
Extraction Method
U
Instrumen t
GC-MS: gas chromatography- mass spectrometry, LC-MS/MS: liquid chromatography-tandem mass spectrometry, LC-UV: liquid chromatography-ultraviolet detector, LC-MS: liquid chromatography-mass spectrometry, CE: capillary electrophoresis, LC-FD: liquid chromatography-fluorescence detector, GC-FID: gas chromatography-flame ionization detection.IL-LLME: Ionic
A
Liquid-Liquid-Liquid Microextraction, SPME: Solid-Phase Microextraction, PP: Protein Precipitation, SDME/MVPE: Single-Drop Microextraction/Micro-Volume Pipette Extraction, DPX: Disposable Pipette Extraction, SPE: Solid-Phase Extraction, DLLME: Dispersive Liquid-Liquid Microextraction.
30
Figure Captions Fig. 1. The total ion current chromatogram of the Flu, Nor, Map, and Paro and their respective derivatives derivative. Fig. 2. The effects of hydrochloric acid concentration (a), the amount of saturated
IP T
NaNO2 (b), derivatization temperature (c), and derivatization time (d) on the peak
Fig. 3. The extraction efficiency of six different solvents.
SC R
area of the four derivatives.
Fig. 4. The selected-ion monitoring mode chromatograms of blank plasma samples
U
and spiked plasma samples (a) and blank urine samples and spiked urine samples (b).
A
CC E
PT
ED
M
A
N
(20 μg L-1 for Flu and Nor, 50 μg L-1 for Map, 100 μg L-1 for Paro).
31
Abundance
Flu-NO
2400000
Nor-NO Map-NO
2000000
Paro-NO
800000
Flu Nor
Map
SC R
1200000
Paro
10.00
14.00
U
400000 6.00
18.00
22.00
N
Time
A
CC E
PT
ED
M
A
Figure 1
32
IP T
1600000
26.00
7x10
5
4x10
5
3x10
5
0.02
0.04
0.06
0.08
6
1x10
6
9x10
5
8x10
5
4x10
5
0.10
0
1.4x10
6
1.2x10
6
1.0x10
6
6.0x10
5
6
1.05x10
6
9.00x10
5
5x10
5
4x10
5
5
0
10
20
30
40
50
60
70
80
90
0
N
4.0x10
1.20x10 P e a k a re a
P e a k a re a
6
A
D erivatization tem perature C
A
CC E
PT
ED
M
Figure 2
100
150
200
A m o u n t o f satu rated N aN O 2 ( L )
Concentration of 100L HCl (mol/L) 1.6x10
50
IP T
8x10
5
1x10
SC R
9x10
5
U
6
P e a k a re a
Peak Area
1x10
33
5
10
15
Derivatization tim e m in
20
1.2x10
6
9.0x10
5
6.0x10
5
3.0x10
5
Flu Nor Map Paro
IP T
6
SC R
Peak aera
1.5x10
0.0
U
e e e ne ene tat orm lfid xen u ha e f l t e u c o o e s i T la m lor n-H nd thy o oro Ch l b E h r Ca Dic
A
CC E
PT
ED
M
A
N
Figure 3
34
(a) Blank and Spiked Plasma Flu-NO
Nor-NO 18000
Abundance 30000
2000 16.50
IP T
16.00
24000
Map-NO
18000
Paro-NO
6000 2000 12.00
14.00
16.00
18.00
20.00
22.00
24.00
U
10.00 Time -->
SC R
12000
N
(b) Blank and Spiked Urine Flu-NO 18000
A
Nor-NO
30000 2000
24000
M
Abundance
16.50
ED
16.00
18000
6000
CC E
2000
PT
12000
Paro-NO
Map-NO
12.00
14.00
16.00
A
10.00 Time -->
Figure 4
35
18.00
20.00
22.00
24.00