Ultra-High Performance Liquid Chromatography (UHPLC) Method for the Determination of Limonene in Sweet Orange (Citrus sinensis) Oil: Implications for Limonene Stability.

Ultra-High Performance Liquid Chromatography (UHPLC) Method for the Determination of Limonene in Sweet Orange (Citrus sinensis) Oil: Implications for Limonene Stability.

Journal of AOAC International (2015-04-11)

Matthew W Bernart

ABSTRACT

The citrus-derived bioactive monoterpene limonene is an important industrial commodity and fragrance constituent. An RP isocratic elution C18 ultra-HPLC (UHPLC) method using a superficially porous stationary phase and photodiode array (PDA) detector has been developed for determining the limonene content of sweet orange (Citrus sinensis) oil. The method is fast with a cycle time of 1.2 min, linear, precise, accurate, specific, and stability indicating, and it satisfies U.S. Pharmacopeia suitability parameters. The method may be useful in its present form for limonene processing, or modified for research on more polar compounds of the terpenome. A forced-degradation experiment showed that limonene is degraded by heat in hydro-ethanolic solution. PDA detection facilitates classification of minor components of the essential oil, including β-myrcene.

MATERIALS

Product Number

Brand

Product Description

PHR1070

Supelco

Dehydrated Alcohol, Pharmaceutical Secondary Standard; Certified Reference Material

62122

Sigma-Aldrich

(R)-(+)-Limonene, technical, ~90% (sum of enantiomers, GC)

62118

Supelco

(R)-(+)-Limonene, analytical standard

183164

Sigma-Aldrich

(R)-(+)-Limonene, 97%

48075

Supelco

Ethanol solution, certified reference material, 2000 μg/mL in methanol

W263303

Sigma-Aldrich

(R)-(+)-Limonene, ≥93%

00590590

(R)-(+)-Limonene, primary reference standard

17236

Sigma-Aldrich

3-(Benzyldimethylammonio)propanesulfonate, BioXtra, ≥99.0% (HPCE)

1012772

USP

Dehydrated Alcohol, United States Pharmacopeia (USP) Reference Standard

PHR1320

Supelco

Residual Solvent - Acetonitrile, Pharmaceutical Secondary Standard; Certified Reference Material

51976

Sigma-Aldrich

Ethanol, BioUltra, for molecular biology, ≥99.8%, (absolute alcohol, without additive, A15 o¹)

733466

Sigma-Aldrich

Acetonitrile, electronic grade, 99.999% trace metals basis

45983

Supelco

Acetonitrile, analytical standard

02851

Sigma-Aldrich

Ethanol, for residue analysis

271004

Sigma-Aldrich

Acetonitrile, anhydrous, 99.8%

PHR1551

Supelco

Acetonitrile, Pharmaceutical Secondary Standard; Certified Reference Material

1601340

USP

Residual Solvent Class 2 - Acetonitrile, United States Pharmacopeia (USP) Reference Standard

493511

Sigma-Aldrich

Ethyl alcohol, Pure, 190 proof, ACS spectrophotometric grade, 95.0%

360457

Sigma-Aldrich

Acetonitrile, ACS reagent, ≥99.5%

34998

Sigma-Aldrich

Acetonitrile, HPLC Plus, ≥99.9%

34851

Sigma-Aldrich

Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%

439134

Sigma-Aldrich

Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%

110086

Sigma-Aldrich

Acetonitrile, ReagentPlus^®, 99%

02882

Sigma-Aldrich

Ethanol, purum, absolute ethanol, denaturated with 2% 2-butanone, A15 MEK¹, ≥99.8% (based on denaturant-free substance)

02856

Sigma-Aldrich

Ethanol, purum, absolute ethanol, denaturated with 4.8% isopropanol, A15 IPA¹, ≥99.8% (based on denaturant-free substance)

02857

Sigma-Aldrich

Ethanol, purum, fine spirit, denaturated with 4.8% methanol, F25 METHYL¹, ~96% (based on denaturant-free substance)

493546

Sigma-Aldrich

Ethyl alcohol, Pure, 200 proof, meets USP testing specifications

494445

Sigma-Aldrich

Acetonitrile, biotech. grade, ≥99.93%

459844

Sigma-Aldrich

Ethyl alcohol, Pure, 200 proof, ACS reagent, ≥99.5%

E7148

Sigma-Aldrich

Ethyl alcohol, Pure, 190 proof, for molecular biology