Application of ultra-sensitive GC-QqQ-MS/MS (MRM) method for the determination of diclofenac in whole blood samples without derivatization

https://doi.org/10.1016/j.jchromb.2021.122860Get rights and content

Highlights

  • A novel GC-QqQ-MS/MS method for determination of diclofenac without derivatization.

  • Liquid-liquid extraction in pH3 with ethyl acetate was applied.

  • Developed method was fully validated.

  • Limit of quantification was 0.1 ng/mL.

  • Method was applied for quantification of diclofenac in human whole blood samples.

Abstract

Diclofenac is one of the most frequently prescribed nonsteroidal anti-inflammatory drugs (NSAID) worldwide. Although it is considered a relatively safe drug, it exhibits high toxicity to some animal populations (e.g., raptors). An ultra-sensitive gas chromatography method, coupled with tandem mass spectrometry (GC-QqQ-MS/MS) with an electron impact (EI) ionization source for diclofenac determination in whole blood samples without a derivatization procedure, was developed and fully validated. Diclofenac-d4 was used as an internal standard. The determination of analytes was performed in the multiple-reaction monitoring (MRM) mode. The method was linear in the range from 0.1 to 200 ng/mL, with a coefficient of determination of 0.999 (R2). The lower limit of quantification was 0.1 ng/mL, and the detection limit was 0.05 ng/mL. The blood samples (200 µL) were prepared by liquid–liquid extraction (pH3) with ethyl acetate. The intra- and interday accuracies and precisions did not exceed 15%. Recovery and matrix effect values were in the range of 92.2–105.9% and −7.8 to 5.9%, respectively. The developed method was applied in authentic blood samples. A simple and precise GC-QqQ-MS/MS method can be potentially applied for routine clinical, toxicological and environmental analysis.

Introduction

Diclofenac is a nonsteroidal anti-inflammatory drug (NSAID) and, similarly to aceclofenac, it is the product of aminophenylacetic acid chemical modification. The pharmacological properties of diclofenac are based on anti-inflammatory, antipyretic and analgesic effects along with inhibition of platelet aggregation. The mechanism of action of this substance is related to the inhibition of cyclooxygenase (COX), which is the enzyme involved in the process of synthesis of prostaglandins from cell membrane lipids. There are two isoforms of cyclooxygenase enzyme: COX-1 (which is activated under physiological conditions in many tissues) and COX-2 (whose activity increases significantly during the inflammatory process) [1]. Diclofenac demonstrates activity on both forms of cyclooxygenase [2], however, the inhibition of COX-2, which results in decreased synthesis of proinflammatory and nociceptive prostaglandins [1], [3], is the basis for the process of the analgesic and anti-inflammatory effects of diclofenac. Diclofenac can be administered orally, rectally, intramuscularly, by intravenous infusion as well as externally (especially as eye drops or a dermis gels, liquids and patches). Dermal application significantly reduces the toxic effects of the drug. Diclofenac is used in the treatment of pain and inflammation, e.g., rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, renal colic and gout attacks as well as menstrual, postoperative and post-traumatic pain disorders. Diclofenac was introduced for therapeutic use in 1974 [1], and now is one of the most frequently prescribed NSAIDs worldwide [4]. Over 7 million people use diclofenac each year [5]. Moreover, diclofenac is contained in the drug Arthrotec®, which is sold on illegal online marketplaces as an abortion pill [6]. Determination of diclofenac in blood samples is useful in terms of veterinary and environmental studies. This substance is widely used as an anti-inflammatory drug in livestock treatment in veterinary medicine. However, diclofenac is highly toxic to birds of prey as well as scavenging birds. Diclofenac poisoning is a major cause of population decline in some vulture species that feed on the meat of animals treated with diclofenac prior to death [7].

Determination of diclofenac in biological samples was performed by the use of high-performance thin-layer liquid chromatography [8] and high-performance liquid chromatography with electrochemical detection [9], fluorimetric detection [10], UV detection [11], [12], [13] and coupled with tandem mass spectrometry [14]. Among gas chromatographic methods, detection of diclofenac was achieved by the use of electron-capture detector [15] flame ionization detector [16], [17], [18], and mass spectrometry [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33].

This paper aims to apply an ultra-sensitive gas chromatography-tandem mass spectrometry with the triple quadrupole (GC-QqQ-MS/MS) method for the determination of diclofenac without derivatization in blood samples. The developed and fully validated method was applied for diclofenac quantification in authentic human whole blood samples.

Section snippets

Chemicals

Water (Chromasolv® LC–MS), methanol (Chromasolv® LC–MS), ethyl acetate, and formic acid were purchased from Sigma-Aldrich (Steinheim, Germany); ammonium chloride from Fluka (Buchs, Switzerland); diclofenac sodium salt was purchased from Sigma-Aldrich (Steinheim, Germany); and diclofenac-d4 (isotopic purity: 98.7%) was purchased from TRC (Toronto, Canada); Standard solutions of diclofenac and internal standard (IS) diclofenac-d4, were prepared in methanol at concentration of 10 mg/mL. The stock

Optimization of mass spectrometer parameters

In order to optimize the mass spectrometry parameters of the method, the analytical standard of diclofenac at a concentration of 100 ng/mL was injected and analyzed in the above described chromatographic conditions in the scan mode in the range of 40–500 m/z. A chromatographic signal, characterized by the spectrum shown in Fig. 1E, was observed at the retention time of 15.19 min. The ion with 277 m/z was selected as the precursor ion. Diclofenac, in the high temperatures of the injection port

Comparison of chromatographic methods in determination of diclofenac

The most selective, sensitive and accurate methods for diclofenac determination in biological samples are liquid and gas chromatographic methods coupled with mass spectrometry. A comparison of gas chromatographic methods with mass spectrometry detection applied for diclofenac quantification is presented in Table 3. Most articles describe the methods with the use of an electron impact (EI) ion source [19], [21], [22], [23], [24], [25], [27], [28], however, De Jong et al. [20] applied chemical

Conclusion

An ultra-sensitive gas chromatographic method coupled with tandem mass spectrometry (GC-QqQ-MS/MS) for diclofenac determination in biological samples without derivatization was evaluated and fully validated. The method described in this paper is selective and precise, while sample preparation is fast and simple. To our knowledge, presented method for diclofenac determination, is the first reported technique which not require the use of any derivatization agents. Sample preparation is based on

Ethical statement

All procedures performed in this study were in accordance with the ethical standards of the national committee and with the 1964 Declaration of Helsinki. Whole blood samples were sent to our institute for toxicological analysis. This article does not contain any studies with living human participants or animals performed by any of the authors.

CRediT authorship contribution statement

Paweł Szpot: Conceptualization, Methodology, Writing – original draft, Visualization. Olga Wachełko: Validation, Writing – original draft. Marcin Zawadzki: Writing - review & editing, Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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