Metabolomics Creative Proteomics

Limonene Analysis Service

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What is Limonene?

Limonene is a naturally occurring compound classified as a monocyclic monoterpene. It is a colorless liquid with a pleasant, citrus-like odor. This compound is commonly found in the peels of citrus fruits, such as oranges, lemons, and limes, but it is also present in various other plants, including conifer trees. The chemical structure of limonene consists of two isomeric forms: D-limonene and L-limonene. These isomers are mirror images of each other, with the D-limonene form being the most prevalent and widely used. Limonene exhibits various biological and chemical properties, making it a fascinating subject of study and analysis.

The demand for limonene analysis arises from its diverse applications across multiple industries. Whether it's in the food and beverage sector, where limonene is used to enhance flavor, or in the cosmetics industry, where it imparts fragrance and therapeutic benefits, knowing the exact composition and concentration of limonene is crucial. Additionally, limonene has demonstrated potential medicinal properties, including anti-inflammatory and anti-cancer effects. Accurate analysis is vital for research and development in the pharmaceutical field.

Creative Proteomics' Limonene Analysis Services

At Creative Proteomics, our limonene analysis services cover a wide array of specific projects related to limonene metabolism analysis. These projects include:

  • Determination of Limonene Content: Accurately quantifying the concentration of limonene in various samples.
  • Identification of Limonene Isomers: Distinguishing between D-limonene and L-limonene, as well as other limonene isomers.
  • Metabolite Profiling: Studying the metabolic products of limonene in different biological systems.
  • Toxicology Studies: Investigating the safety and potential toxicological effects of limonene and its derivatives.
  • Pharmacokinetic Analysis: Understanding how limonene is absorbed, distributed, metabolized, and eliminated in the body.

Limonene Analytical Techniques

Our limonene analysis services are built upon advanced analytical techniques, with a primary focus on mass spectrometry. We employ state-of-the-art instruments to ensure the highest level of precision. Some of the key instruments utilized in our limonene analysis include:

  • Gas Chromatography-Mass Spectrometry (GC-MS): This technique is the cornerstone of limonene analysis. We utilize the Agilent 7890B GC system coupled with the Agilent 5977A Mass Selective Detector (MSD) to separate and quantify limonene in complex mixtures, providing accurate data on its presence and concentration.
  • Liquid Chromatography-Mass Spectrometry (LC-MS): For applications requiring liquid-phase analysis, we utilize the Thermo Fisher Scientific Q Exactive™ LC-MS system, which offers a highly sensitive and selective method for limonene analysis.
  • High-Performance Liquid Chromatography (HPLC): HPLC is used to separate limonene isomers efficiently and quantify their individual concentrations.

List of Analyzed Limonenes, including but not limited to

Creative Proteomics offers comprehensive limonene analysis services, not limited to D-limonene but extending to various limonene derivatives and isomers. Here is a list of some limonene variants we can analyze:

D-limoneneL-limonenealpha-Pinenebeta-PineneLimonene oxide
CarveolPerillyl alcoholMyrceneGamma-terpinenep-Cymene

Sample Requirements for Limonene Assay

Sample TypeSample AmountContainer Type
Essential Oils1-2 mL (minimum)Amber glass vial with a screw cap
Fruit Peel Extracts1-2 g (minimum)Airtight, labeled containers
Biological SamplesAs required for analysisAppropriate tubes or containers
Other MatricesSpecify the amountContainers based on sample type

Applications of Limonene Qualitative and Quantitative Analysis

Pharmaceutical Research and Drug Development: Limonene, with its potential anti-inflammatory and anti-cancer properties, is a crucial component in the development of pharmaceuticals. Our analysis services facilitate the study of limonene's therapeutic potential and its formulation for various medical conditions.

Toxicological Studies: Evaluating the safety of pharmaceutical products and potential interactions with biological systems is a core application. Our precise analysis plays a pivotal role in toxicological studies, ensuring product safety.

Pharmacokinetics: Understanding how limonene is metabolized, distributed, and excreted in the body is vital for drug efficacy. Our services support pharmacokinetic studies, improving the understanding of drug behavior.

Metabolite Profiling: Limonene's effects within biological systems are explored through the study of its metabolic products. This knowledge aids in understanding its influence on the human body.

Flavor and Fragrance Industry: Limonene's pleasant citrus aroma makes it a key ingredient in perfumes, air fresheners, and food flavorings. Our analysis services ensure that the quality and consistency of products meet industry standards.

Food and Beverage Industry: Limonene is used as a natural flavor enhancer in the food and beverage sector. Accurate analysis is essential for regulatory compliance and maintaining product quality.

Cosmetics and Personal Care: The cosmetics and personal care industry incorporates limonene for its pleasant scent and potential therapeutic benefits. Our analysis ensures product safety and quality.

Environmental Monitoring: Limonene analysis extends to environmental assessments. Monitoring its presence in industrial emissions is essential for environmental impact assessments and safety compliance.

Research and Development: Researchers and scientists rely on accurate limonene analysis to innovate and create new products. Our services support innovation and product development.

Nutraceuticals and Dietary Supplements: Limonene is found in various dietary supplements and nutraceuticals. Qualitative and quantitative analysis ensures product quality and efficacy in promoting health.

If you would like to analyze other terpenes or learn more, please contact us. We look forward to working with you.

Case: Revealing the Pharmacological Promise of Inhalable (+)-Limonene through GC–MS Analysis


Essential oils, known for their traditional use in promoting relaxation, have been explored for their anxiolytic effects. This study focuses on the anxiolytic potential of (+)-limonene, a key component in citrus oils, administered via inhalation. It aims to investigate this potential and understand the underlying mechanisms, offering insights into its application in aromatherapy.


Male Swiss albino mice (25–35 g) were used as the experimental subjects. The study conducted its experiments under controlled environmental conditions with appropriate ethical approvals.

Technical Methods

The gas chromatography analysis was a central component of this study and was conducted as follows:

  • Sample Collection: The study involved collecting air samples at specific time intervals to measure the concentration of (+)-limonene. Sampling points included 13 minutes, 23 minutes, and 33 minutes. Additionally, concentrations at 7 minutes and 14 minutes were calculated based on a linear equation established using standard limonene concentration data.
  • Linear Equation Establishment: The data obtained from the analysis were used to create a linear equation: y = 144098x − 137498 with an excellent correlation coefficient (r^2) of 0.9996. This equation allowed for the calculation of limonene concentrations at various sampling times.
  • Analytical Parameters: The analytical parameters were presented in Table 1. These parameters included the time of collection, area, and limonene concentration (measured in μg/cm³) at each sampling point, providing a detailed insight into the kinetics of dispersion of (+)-limonene.
  • Volatility Assessment: The results of the Gas Chromatography Analysis indicated that the concentration of (+)-limonene increased over time, reaching 7.56 μg/cm³ of air at 33 minutes. This highlighted the high volatility of (+)-limonene and its ability to disperse into the surrounding environment.


The Gas Chromatography-Mass Spectrometry (GC–MS) analysis was a key aspect of this study, offering valuable insights into the concentration and volatility of (+)-limonene. The results from the GC–MS analysis are summarized as follows:

  • Linearity Curves: The study constructed two essential linearity curves. The first curve correlated peak areas with the range of limonene concentrations, revealing a linear equation: y = 144098x − 137498, with a high correlation coefficient (r^2) of 0.9996. This equation facilitated the calculation of limonene concentrations at various sampling times. The second curve established a relationship between the estimated concentration of the samples and the time of sample collection, yielding a linear equation: y = 0.1895x + 1.3915 with an r^2 of 0.9940.
  • Sample Analysis: The study injected 50 μL of (+)-limonene into the enclosed system for GC–MS analysis. Aliquots of 10 μL were collected at specific time points (13 min, 23 min, and 33 min) from the atmosphere within the system. The results of the analysis provided insights into the time-dependent concentration of (+)-limonene in the enclosed environment.
  • Identification of Compound: The GC–MS analysis was performed using a Shimadzu GC–MS-QP5050A system. It identified (+)-limonene by comparing its GC retention index to n-alkanes (C8–C20) and its mass spectra with the Wiley 275.L database.


  1. Lima, Naiana GPB, et al. "Anxiolytic-like activity and GC–MS analysis of (R)-(+)-limonene fragrance, a natural compound found in foods and plants." Pharmacology Biochemistry and Behavior 103.3 (2013): 450-454.
For Research Use Only. Not for use in diagnostic procedures.


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