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

576956

Sigma-Aldrich

Acetonitrile solution, contains 0.1 % (v/v) formic acid, suitable for HPLC

62122

Sigma-Aldrich

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

574732

Sigma-Aldrich

Acetonitrile solution, contains 0.1 % (v/v) trifluoroacetic acid, suitable for HPLC

48075

Supelco

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

183164

Sigma-Aldrich

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

685461

Sigma-Aldrich

Acetonitrile solution, contains 10.0% acetone, 0.05% formic acid, 40.0% 2-propanol

574724

Sigma-Aldrich

Acetonitrile solution, contains 0.05 % (v/v) trifluoroacetic acid

62118

Supelco

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

E2385

Supelco

Ethanol standards 10% (v/v), 10 % (v/v) in H₂O, analytical standard

51976

Sigma-Aldrich

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

17236

Sigma-Aldrich

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

W263303

Sigma-Aldrich

(R)-(+)-Limonene, ≥93%

PHR1070

Supelco

Dehydrated Alcohol, Pharmaceutical Secondary Standard; Certified Reference Material

1012772

USP

Dehydrated Alcohol, United States Pharmacopeia (USP) Reference Standard

00590590

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

679186

Sigma-Aldrich

Acetonitrile solution, contains 0.05 % (w/v) ammonium formate, 5 % (v/v) water, 0.1 % (v/v) formic acid, suitable for HPLC

PHR1320

Supelco

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

271004

Sigma-Aldrich

Acetonitrile, anhydrous, 99.8%

493511

Sigma-Aldrich

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

437557

Sigma-Aldrich

Acetonitrile, ≥99.5%, ACS reagent

493538

Sigma-Aldrich

Ethyl alcohol, Pure, 190 proof, meets USP testing specifications

50387

Supelco

Acetonitrile, suitable for HPLC, gradient grade, ≥99.9% (GC)

459836

Sigma-Aldrich

Ethyl alcohol, Pure, 200 proof, anhydrous, ≥99.5%

733466

Sigma-Aldrich

Acetonitrile, electronic grade, 99.999% trace metals basis

45983

Supelco

Acetonitrile, analytical standard

675415

Sigma-Aldrich

Acetonitrile, HPLC Plus, ≥99.9%, poly-coated bottles

PHR1551

Supelco

Acetonitrile, Pharmaceutical Secondary Standard; Certified Reference Material

1601340

USP

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