Analytical Method Validation for Volatile Compounds

The analytical method was validated according to the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Q2(R2) guidelines [38 ]. Six calibration standards (Cal) and three quality control (QC) samples at different concentrations (QC_high, QC_med, and QC_low) were prepared by serial dilution of separate stock solutions containing 7.3 mM (−)-β-pinene, 88.1 mM (+)-α-pinene, 7.3 mM α-terpinene, 4.9 mM β-caryophyllene, 5.2 mM β-damascone, 7.3 mM camphene, 6.6 mM citral, 128.0 mM citronellol, 5.0 mM citronellyl acetate, 105.1 mM β-damascenone, 7.3 mM limonene, 12.2 mM eugenol, 11.2 mM methyleugenol, 9.0 mM farnesol, 129.7 mM geraniol, 10.2 mM geranyl acetate, 19.4 mM linalool, 64.8 mM nerol, 10.2 mM neryl acetate, 7.5 mM p-cymene, 7.5 mM phenylethanol and 25.9 mM rose oxide in n-heptane. Cis-3-hexen-1-ol was added as an internal standard (IS) with a final concentration of 10 mM to each Cal and QC. For accuracy and precision Cal and QC samples were analysed on 5 separate days over a course of 2 weeks. QC samples were measured in duplicates. Accuracy was determined as a bias; intra-day and inter-day imprecision were calculated as the relative standard deviation (RSD_R and RSD_T, respectively) [39 ]. Specificity was demonstrated by spiking authentic rose oil samples and four different EO samples with the analytes at intermediate levels of the calibration range. A further spiking experiment was conducted by adding QC_high and QC_low level concentrations to an authentic rose oil sample. The recovery effect (RE) was determined for the spiking experiments (Formula (1)) [40 (link)]. Retention index (RI) was determined by analysing a homologous series of n-alkanes (C8–C20, C21–C40, analytical standard) purchased from Sigma Aldrich (St. Louis, MO, USA). The RI was calculated according to the van den Dool and Kratz equation [41 (link)]. Peak resolution (Rs) was determined according to European Pharmacopeia (Ph. Eur.) 11.2 [42 ]. Limit of detection (LoD) and limit of quantification (LoQ) were calculated based on the standard deviation of the response and the slope of the calibration curve [38 ]. The method was tested for robustness by changing the split ratio, flow, detector temperature, inlet temperature and temperature ramp of the established methods. Furthermore, the method was transferred to different instruments.
$$RE [\%]=\frac{con{c}_{experimental spiked sample}-con{c}_{experimental sample}}{con{c}_{spiking }}\times 100$$