Documentation of Liquid Chromatography-Mass Spectrometry (LC-MS) Technique for Quantitative Analysis of Naproxen in Human Plasma

Abstract

Naproxen is a commonly used non steroidal anti-inflammatory drug (NSAID) employed in treatment of pain, fever and inflammation. It has the ability to bind and inhibit synthesis of prostaglandins and produces anti inflammatory effect. Accurate quantification of naproxen in human plasma is essential for bioavailability, pharmacokinetic and clinical studies. Liquid chromatography -Mass Spectrometry has emerged as a gold standard analytical approach for such purpose due to its good sensitivity, specificity and rapid analysis. A quick and selective LC-MS approach for quantification of naproxen in human plasma can be used in therapeutic drug monitoring as well as in its bioequivalence study. This review article highlights instrumentation, process and methodology employed in quantitative analysis of naproxen in human plasma using LC-MS. Factors such as sample preparation technique (for example, protein precipitation, liquid-liquid extraction and solid-phase extraction), chromatographic conditions, mass spectrometric parameters and method validation are critically important.

Keywords

Introduction

Liquid Chromatography -Mass Spectrometry Technique

Liquid chromatography- mass spectrometry LC-MS is an  analytical chemistry technique that combines the physical separation capabilities of liquid chromatography or HPLC with the mass analysis capabilities of mass spectrometry [9]. Coupled chromatography systems are common in chemical analysis because the individual capabilities of each technique are enhanced synergistically when combined [10]. While liquid chromatography separate mixtures with multiple components, mass spectrometry provides spectral information that can be used to identify each separated component [16]. LC-MS is not only sensitive but provides selective detection reducing need for complete chromatographic separation [17]. LC-MS is also ideal for metabolomics due to its broad coverage for a wide range of chemicals [18]. This Tandem approach can be used to investigate biochemical, organic and inorganic compounds often encountered in complex samples of environmental and biological origin. Hence, LC-MS can be used in variety of industries including biotechnology, environment monitoring ,food processing and pharmaceutical agrochemical and cosmetics [19]. In addition to the liquid chromatography and mass spectrometry devices, LC-MS system includes an interface for efficiently transferring the separated component from the LC column into the MS ion source. The interface is required since the LC and MS devices are inherently incompatible. While the mobile phase in a LC system is a pressurized liquid, the MS analyzers typically operate under high vacuum. Therefore, it is not possible to directly pump the eluate from the LC column into the MS source. The interface is a mechanically simple element of the LC-MS system that conveyed the maximum quantity of analyte, remove a significant portion of the mobile phase used in LC and preserves the chemical identity of the chromatography products. The interface should not interfere with the ionizing efficiency and vacuum conditions of the MS system [19].

Instrumentation of Liquid Chromatography -Mass Spectrometry (LC-MS)

LC-MS system consist of a pump, an injector to introduce the sample, a column to separate the analytes and a mass spectrometer detector. Liquid chromatography separates dissolved analytes by utilizing variations in the respective affinity for the solvent or mobile phase and that of stationery phase or column media. Separation can be influenced by changing such variable as column packing material, solvent choice, solvent ratios and pH. Most modern LC systems have at least two different solvents: an organic phase (usually methanol or acetonitrile) and an aqueous buffer at a specific pH. Introduction of the analytes into the MS detector can be achieved through several different systems including electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). In most cases the eluent needs to be removed before ionization and introduction into the mass spectrometer [11].

Applications of LC-MS

LC-MS allows detection of low concentration of naproxen, which is critical in pharmacokinetic investigations. Human plasma contains several components and LC-MS can successfully distinguish naproxen from these interferences. Mass spectrometry can analyze, identify and quantify both naproxen and its metabolites, giving a complete view of drug metabolism. Hence it makes LC-MS preferred choice for analysis of naproxen in clinical and research settings. LC-MS play a crucial role in metabolomics and proteomics as well as identifying proteins in biological systems. The coupling of MS with LC systems is effective because liquid chromatography can separate delicate and complex natural mixtures, whose chemical composition needs to be accurately determined. LC-MS has applications in volatile explosive residue analysis.[20]. Other important applications of LC-MS include the analysis of food, pesticides, and plant phenols [21].

Aim and purpose

1. Pharmacokinetic studies: These studies aim understand how naproxen is absorb, distributed, metabolised and  eliminated in the body. This aids in determining dosing schedule and understanding the duration of action.
2.  Therapeutic drug monitoring: For patients using naproxen, monitoring plasma level insure the drug remain within therapeutic  range, optimizing efficacy and minimizing side effects and toxicity.
3. Bioavailability and bio equivalence studies: In the development of new formulation or generic drug, LC-MS analysis of naproxen in plasma evaluates how much of the drug reaches systemic circulation and compares bioavailability
4. Toxicology and safety study: To detect build up in the plasma which could be associated with toxicity particularly in long-term or high-dose therapy [31].
5.  Drug interaction studies: By analyzing plasma level in patients taking multiple medications, researchers can detect interactions that affect naproxen’s pharmacokinetics [22].

Why Zidovudine is chosen as standard?

Zidovudine is chosen as benchmark because it has similar physicochemical properties to Naproxen. Zidovudine has similar chromatographic behavior and ionization property in the mass spectrometer. Zidovudine has define retention times and mass spectral fragmentation patterns that can aid in developing sensitive and specific methods for detecting and quantifying naproxen in human plasma. In addition to Zidovudine, several other drugs may be used as standards for analyzing naproxen in human plasma via LC-MS

Ibuprofen: Another non-steroidal anti-inflammatory drug that may have similar properties to naproxen.

Diclofenac: A commonly used non-steroidal anti-inflammatory drug with distinct pharmacokinetic properties.

Caffeine: Sometimes used due to its well characterized pharmacokinetic and broad clinical relevance.

Warfarin: Used sometimes in pharmacokinetic studies specially in the context of drug interaction research.

Alternative methods for analysis of Naproxen

While liquid chromatography mass spectrometry (LC-MS) is a very sensitive and specific technique for detecting naproxen in human plasma, various alternative methods can be utilized depending on the required sensitivity, specificity and instrumentation availability. Some of the common alternatives are:

1. High performance liquid chromatography (HPLC) with UV detection:  This is a widely used alternative for naproxen quantification. Naproxen has high absorbance in the UV range, which makes UV detection effective. However, HPLC -UV may lack the sensitivity and specificity of LC-MS.
2. Gas chromatography-Mass spectrometry (GC-MS): GC-MS can we used for naproxen analysis, however it requires a derivatization step to make naproxen volatile for GC analysis. This method is less commonly used for naproxen because of the additional sample preparation steps required.
3. Capillary Electrophoresis (CE): CE is an alternative method that can separate ionic compounds based on their charge and size. It is relatively fast and requires minimal solvent, making it environmentally friendly. However, it may not offer the same level of sensitivity as LC-MS.
4. Immunoassays: Techniques like enzyme- linked immunosorbent assay (ELISA) can be performed to quantify naproxen. Immunoassays are generally highly specific, but they can exhibit cross- reactivity with structurally related compounds and may lack quantitative precision.
5. Ultra-Performance Liquid Chromatography (UPLC): UPLC is an upgraded variant of HPLC that enables faster analysis with improved resolution and sensitivity. When combined with UV or fluorescence detection, it can serve as an alternative to traditional HPLC-UV, providing increased efficiency.
6.  Fluorescence Spectroscopy: This method involve derivatizing naproxen to form a fluorescent molecule, allowing for detection via fluorescence. However, it involves additional chemical processes for derivatization and maybe less specific than LC-MS
7. Electrochemical Detection (ECD): Electrochemical detection can be used with HPLC to detect naproxen based on its electroactive properties. This method can be highly sensitive but is typically used less frequently than other options. [33]

Each technique has unique strengths and weaknesses in terms of sensitivity, specificity, cost and throughput. LC-MS remains the most reliable approach for naproxen analysis in human plasma due to its high sensitivity and specificity, but the alternatives maybe chosen depending on analytical needs and equipment availability. LC-MS is commonly accepted by regulatory agencies ( like FDA and EMA) for drug quantification in bio analytical studies because of its reliability. For clinical studies, LC-MS is typically considered as  the ‘gold standard’ for ensuring data quality and reproducibility.

Process for analyzing naproxen in human using LC-MS technique

Analyzing naproxen in human plasma by LC-MS generally involves several steps:

1. Sample collection and preparation
2. Method development
3. LC-MS setup
4. Calibration, validation and analysis

Sample collection and storage: Collect the blood samples from participants and immediately process them to separate plasma by centrifuging the blood sample at 2000-3000 rpm for 10 minutes

Sample preparation: Naproxen is extracted from plasma using protein precipitation for liquid- liquid extraction process. Alternatively, for a cleaner extract, contaminants can be removed using solid phase  extraction (SPE).

LC-MS method Development:

For Liquid chromatography (LC),

Column: Use a reverse – phase column, typically C18 for optimal separation. Mobile phase: A gradient or isocratic elution with mobile phases such as water with 0.1% formic acid (for aqueous phase) and acetonitrile or methanol with 0.1% formic acid (for organic phase) is most common. The addition of formic acid helps to improve ionization and peak shape.

Flow Rate:  Set an appropriate and acceptable flow rate based on column specification and technique sensitivity requirements.

For Mass Spectrometry (MS),

Ionization: Electrospray ionization (ESI) is commonly used in LC-MS analysis of Naproxen, as it  generates charged molecules from the analyte in the solution.

Mass Analysis: In the mass spectrometer, naproxen ions are separated based on their mass-to-charge ratio. The MS is set to detect naproxen’s specific m/z value, which is known and helps in identifying the compound.

Quantification: Multiple Reaction Monitoring (MRM) is a technique often used for quantification, where specific precursor product ion transitions are monitored, enhancing sensitivity and selectivity.[36]

CONCLUSION

Naproxen remains one of the most often used NSAIDs due to its effectiveness in treating pain, inflammation and fever across a number of conditions. An alternative HPLC method for quantifying naproxen in human plasma has been successfully studied. A simple and inexpensive liquid -liquid extraction process combined with an isocratic chromatography condition employing a reverse phase column results in an assay that is ideally suited for real time analysis. The analysis of Naproxen in human plasma using LC-MS with zidovudine as an internal standard is a reliable method for quantifying drug levels enabling for effective therapeutic monitoring, bioequivalence and pharmacokinetic investigations.

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