Abstract
Kashmir saffron (Crocus sativus L.), also known as Indian saffron, is an important Asian medicinal plant with protective therapeutic applications in brain health. The main bioactive in Kashmir or Indian Saffron (KCS) and its extract (CSE) are apocarotenoids picrocrocin (PIC) and safranal (SAF) with carotenoids, crocetin esters (crocins), and crocetins. The ultra-fast liquid chromatography(UFLC)- photodiode array standardization confirmed the presence of biomarkers PIC, trans-4-GG-crocin (T4C), trans-3-Gg-crocin (T3C), cis-4-GG-crocin (C4C), trans-2-gg-crocin (T2C), trans-crocetin (TCT), and SAF in CSE. This study’s objectives were to develop and validate a sensitive and rapid UFLC-tandem mass spectrometry method for PIC and SAF along T4C and TCT in rat plasma with internal standards (IS). The calibration curves were linear (R2 > 0.990), with the lower limit of quantification (LLOQ) as 10 ng/mL. The UFLC-MS/MS assay-based precision (RSD, <15%) and accuracy (RE, −11.03–9.96) on analytical quality control (QC) levels were well within the acceptance criteria with excellent recoveries (91.18–106.86%) in plasma samples. The method was applied to investigate the in vivo pharmacokinetic parameters after oral administration of 40 mg/kg CSE in the rats (n = 6). The active metabolite TCT and T4C, PIC, SAF were quantified for the first time with T3C, C4C, T2C by this validated bioanalytical method, which will be useful for preclinical/clinical trials of CSE as a potential neuroprotective dietary supplement.
Highlights
The specificity and selectivity were investigated by comparing the retention times in chromatograms of blank plasma added with analytes and blank plasma, and plasma sample after oral administration of Compounds of Kashmir saffron extract (CSE); no interference peak was observed at a retention time of analytes of endogenous substances in the plasma samples. (Figure 3) The mean signal-to-noise ratios for T4C, TCT, PIC, SAF, IS1, and IS2 were 18.90 ± 4.28, 24.97 ± 4.82, 17.02 ± 3.39, 34.70 ± 7.08, 77.72 ± 8.28, 88.69 ± 10.77, respectively
Dilution integrity determined by analyte spiking above the upper limit of quantification (ULOQ) and diluted with blank plasma brought into the calibration curve was analyzed to obtain acceptable concentration after proper dilution with blank plasma
The results showed that the precision and accuracy of the diluted quality control (QC) were within the 15%
Summary
Mass Spectrometry (UFLC-MS/MS): In Vivo Pharmacokinetics of Apocarotenoids and Carotenoids. This saffron mainly grows in India’s Kashmir region, known as Kashmir or Indian Saffron (KCS) This KCS is rich in bioactive constituents, carotenoids (crocetin esters, i.e., crocins, crocetin), and apocarotenoids such as picrocrocin (PIC) with safranal (SAF) (Figure 1) [1,2,3,4]. The present study aimed to develop and validate a sensitive and short UFLC-MS/MS bioanalytical method for the simultaneous determination of apocarotenoids (PIC, SAF) and carotenoids (T4C, TCT) in the rat plasma. This methodology was further validated with internal standards reserpine and chloramphenicol as per the USFDA (United States Food and Drug Administration)and. According to the phytopharmaceutical guidelines of the Drugs Controller General (DCG) (India) and other regulations, this bioanalytical method will be useful for preclinical or phase 1 clinical trials
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