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

17236

Sigma-Aldrich

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

62118

Supelco

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

62122

Sigma-Aldrich

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

1012772

USP

Dehydrated Alcohol, United States Pharmacopeia (USP) Reference Standard

E7148

Sigma-Aldrich

Ethyl alcohol, Pure 190 proof, for molecular biology

183164

Sigma-Aldrich

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

W263303

Sigma-Aldrich

(R)-(+)-Limonene, ≥93%

574732

Sigma-Aldrich

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

574724

Sigma-Aldrich

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

685461

Sigma-Aldrich

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

48075

Supelco

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

576956

Sigma-Aldrich

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

PHR1320

Supelco

Residual Solvent - Acetonitrile(solution in DMSO), Pharmaceutical Secondary Standard; Certified Reference Material

00590590

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

PHR1551

Supelco

Acetonitrile(Neat), Pharmaceutical Secondary Standard; Certified Reference Material

493511

Sigma-Aldrich

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

493538

Sigma-Aldrich

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

271004

Sigma-Aldrich

Acetonitrile, anhydrous, 99.8%

437557

Sigma-Aldrich

Acetonitrile, ≥99.5%, ACS reagent

459836

Sigma-Aldrich

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

40347

Sigma-Aldrich

Ethanol, absolute, sales not in Germany, ≥99.8% (vol.)

792829

Sigma-Aldrich

Ethyl alcohol, Pure, 140 proof, meets water USP testing specifications, Excise Tax-free, Permit for use required

792799

Sigma-Aldrich

Ethyl alcohol, Pure, 190 proof, ACS reagent, meets USP testing specifications, Excise Tax-free, Permit for use required

L010300

Sigma-Aldrich

Ultrapure Acetonitrile

V800024

Sigma-Aldrich

Acetonitrile, AR, ≥99.5%

51976

Sigma-Aldrich

Ethanol, BioUltra, Molecular Biology, ≥99.8%, (absolute alcohol, without additive, A15 o¹)

792810

Sigma-Aldrich

Ethyl alcohol, Pure, 140 proof, Excise Tax-free, Permit for use required

1601340

USP

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

733466

Sigma-Aldrich

Acetonitrile, electronic grade, 99.999% trace metals basis

675415

Sigma-Aldrich

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