Quantitative Determination and Characterization of a Kashmir Saffron (Crocus sativus L.)-Based Botanical Supplement Using Single-Laboratory Validation Study by HPLC-PDA with LC-MS/MS and HPTLC Investigations

Abstract

Food ingredients hold a higher nutritional value as a botanical supplement playing a vital role in modifying and maintaining the physiological conditions that improve human health benefits. The Kashmir saffron (Crocus sativus L; KCS) obtained from dried stigmas is known for its aroma precursors and apocarotenoid derivatives, imparting a wide range of medicinal values and therapeutic benefits. In the present study, a simultaneous determination of apocarotenoids and flavonoids in stigma-based botanical supplements was carried out using analytical investigations. The high-performance thin-layer chromatography-based qualitative analysis of the raw material (stigmas, stamens, and tepals) and stigma extract has been carried out to identify apocarotenoids and flavonoids. The rapid HPLC-PDA method for the simultaneous quantification of KCS apocarotenoids was robust, precise (<5.0%), linear (R ² > 0.99), and accurate (80-110%) as per the single-laboratory validation data. Furthermore, the combined-expanded uncertainty (95%; K = 2) was calculated and found as 0.0035-0.007% (<5.0%) as per the EURACHEM guide for this HPLC analysis. Additionally, an untargeted identification of 36 compounds in the botanical supplement was based on the elution order, UV-vis spectra, mass fragmentation pattern, and standards by ESI-MS/MS analysis with comprehensive chromatographic fingerprinting. Thus, these analytical approaches enable a composite profile of the stigma-based extract as a potential supplement for human health benefits.

Figure 1

Kashmir saffron (C. sativus L.). Habit (A), local gradings of Kashmir saffron (B), dried stigmas (C), stamens (D), and tepals (E).

Figure 2

Structure of apocarotenoids and flavonoids from Kashmir saffron (C. sativus L.). Picrocrocin (1), trans-4-GG-crocin (2), trans-3-Gg-crocin (3), cis-4-GG-crocin (4), trans-2-gg-crocin (5), trans-crocetin (6), safranal (7), kaempferol-3-O-β-sophoroside (8), quercetin-3-O-sophoroside (9), and quercetin-3,4′-di-O-glucoside (10).

Figure 3

HPTLC fingerprinting of flower parts of Kashmir saffron (KCS) under UV 254 nm (A), under 366 nm (B), under white light after derivatization (C), and under UV 366 nm after derivatization (D); track details: T1–T6: reference standards, where, picrocrocin (1), trans-4-GG-crocin (2), trans-3-Gg-crocin (3), kaempferol-3-O-β-sophoroside (8), kaempferol-O-glucoside (9), and quercetin-3,4′ di-O-glucoside (10); T7–T10: the stigma of KCS; T11–T14: stamens of KCS; and T15–T18: tepals of KCS.

Figure 4

Kashmir saffron (C. sativus L.)-based botanical supplement (CSE) standardization by batch analysis (n = 3) by HPLC-PDA analysis. Representative chromatogram at wavelengths of 250 nm (compound 1), 440 nm (compound 2–6), and 320 nm (compound 7).

Figure 5

HPTLC fingerprinting of the stigma-based botanical supplement from KCS (n = 10) with reference standards under UV 254 nm (A), under UV 366 nm (B), under white light after derivatization (C), and under 366 nm after derivatization (D). Track details: T1–T4: reference standards, where picrocrocin (1), trans-4-GG-crocin (2), trans-3-Gg-crocin (3), and kaempferol-3-O-β-sophoroside (8), and T5–T14: samples of stigma-based botanical supplements (CSE).

Figure 6

LC–MS/MS TIC of 36 compounds from apocarotenoids and flavonoids from KCS and its botanical supplements in the positive and negative mode.

Figure 7

Positive and negative ESI-MS/MS spectra highlighting the main fragments of detected peaks as (AP17) picrocrocin and (AP36) safranal (A,B) with (CR22) trans-4-GG crocin, (CR24) trans-3-Gg crocin, (CR28) cis-4-GG crocin, (CR29) trans-2-gg crocin, and (CR35) trans-crocetin (C–G), respectively. In the case of these apocarotenoids, the fragmentation patterns were in accordance with those proposed by refs (26) and (30).