Acta agriculturae Slovenica, 118/3, 1–9, Ljubljana 2022 doi:10.14720/aas.2022.118.3.2333 Original research article / izvirni znanstveni članek Quantification of apocarotenoids in commercial Indian (Kashmiri) saf- fron using UV-Vis spectroscopy and HPLC analysis Tahir ul Gani MIR 1, Jaskaran SINGH 2, Saurabh SHUKLA 1, 3 Received September 05, 2021; accepted July 10, 2022. Delo je prispelo 5. septembra 2021, sprejeto 10. julija 2022 1 Department of Forensic Science, School of Bioengineering & Biosciences, Lovely Professional University, Phagwara, India 2 Department of Forensic Science, Geeta University, Naultha, Panipat, India 3 Corresponding author, e-mail: sau47bt@gmail.com Quantification of apocarotenoids in commercial Indian (Kashmiri) saffron using UV-Vis spectroscopy and HPLC analysis Abstract: Saffron is considered as the most expensive spice in the world. Due to low production, high demand and high cost, saffron is very prone to adulteration for economic benefits while putting public health at risk. The most significant characteristic for determining the quality of the saffron is color- ing strength (crocin content), which is determined by measur- ing UV-Vis absorption at 440 nm in the aqueous preparations of this spice. Picrocrocin and safranal are other key compo- nents used to determine saffron quality. This article aims to examine the quality of commercial saffron obtained from vari- ous geographical locations of Kashmir (India) by determining their apocarotenoid content using UV-Vis spectrophotometry followed by high-performance liquid chromatography (HPLC) to determine the concentration of saffron metabolites (crocin, picrocrocin and safranal). A total of 31 samples from different origins were used in this study. The UV-Vis spectrophotometric results showed that among 31, only 14 samples fell into grade I, while 9 samples fell in grade II and 5 samples fell in grade III of the ISO category. The remaining 3 samples could not satisfy ISO standards, which indicates that these samples were adulter- ated. The determination of apocarotenoid content using HPLC analysis varied significantly among samples. These variations may be due to different drying and storage conditions or adul- teration. Key words: saffron; adulteration; crocin; safranal; picro- crocin; UV-Vis spectroscopy; HPLC Količinsko ovrednotenje apokarotenoidov v komercialnih vzorcih indijskega (kašmirskega) žafrana z analizo UV-Vis spektroskopije in HPLC Izvleček: Žafran velja za najdražjo začimbo v svetovnem merilu. Zaradi majhne pridelave, velikega povpraševanja in visoke cene je zaradi ekonomskih koristi podvržen ponareja- nju, kar povzroča zdravstvena tveganja. Najznačilnejša lastnost za določanje kakovosti žafrana je njegova sposobnost obarvanja (vsebnost krocina), ki se določa z merjenjem UV-Vis absorb- cije pri 440 nm v vodnih pripravkih te začimbe. Pikrokrocin in safranal sta ostali klučni komponenti, ki se uporabljata za določanje kakovosti žafrana. V raziskavi smo preučevali kako- vost tržnega žafrana pridobljena iz različnih geografskih obmo- čij Kašmirja (Indija) z določanjem vsebnosti apokarotenoidov z UV-Vis spektroskopijo, ki ji je sledila analiza z visokotlačno tekočinsko kromatografijo (HPLC), kjer smo v vzorcih žafrana določali koncentracije metabolitov kot so krocin, pikrokrocin in safranal. V raziskavi je bilo analiziranih 31 vzorcev različ- nega izvora. Rezultati analize z UV-Vis spektroskopijo so po- kazali, da se je med 31 vzorci samo 14 uvrstilo v kvaliteto I, 9 vzorcev seje uvrstilo v kvaliteto II in 5 vzorcev v kvaliteto III, glede na ISO kategorije. Preostali 3 vzorci niso izpolnjevali ISO standardov, kar kaže, da so bili ponarejeni. Vsebnost apokaro- tenoidov v vzorcih se je pri analizi s HPLC značilno razlikovala, kar bi lahko bila posledica različnega sušenja, shranjevanja ali ponarejanja. Ključne besede: žafran; ponarejanje; krocin; safranal; pi- krokrocin; UV-Vis spektroskopija; HPLC Acta agriculturae Slovenica, 118/3 – 20222 T. G. MIR et al. 1 INTRODUCTION Saffron, often referred to as red gold, is obtained from the stigma of Crocus sativus L. (Saxena, 2010). Cro- cus sativus is an angiosperm plant, member of the As- paragales family. The flower of Crocus sativus is solitary, purple, with six petals, three stamens, one style, and three reddish-orange stigmas. Saffron crocus grows through- out the Mediterranean–Europe, and Western Asia. It is commonly cultivated in Iran, Greece, Spain, Italy, Af- ghanistan and India (Kashmir) (Kahriz, 2020). Saffron is quite costly because it is used mainly as a flavoring and aromatizing agent (Mir et al., 2022a; Mzabri et al., 2019). Saffron contains approximately 300 volatile and non- volatile metabolites, including crocin, safranal, picro- crocin, monoterpenes, aldehydes, and various other ca- rotenoids of therapeutic potential (Pandita, 2021). It is a well-known spice that is used to cure a variety of medical conditions, including depression (Siddiqui et al., 2018), cardiovascular illness (Kamalipour & Akhondzadeh, 2011), menstrual irregularities (Beiranvand et al., 2016), asthma (Zilaee et al., 2019)lipid profiles, basophils, eo- sinophils and clinical symptoms in patients with allergic asthma.\nSTUDY DESIGN: Our study was a clinical trial.\nMETHODS: Subjects (N = 80, 32 women and 48 men, 41.25 ± 9.87 years old, insomnia (Taherzadeh et al., 2020), and digestive problems (Khorasany & Hosseinza- deh, 2016). Crocin is a carotenoid chemical compound responsible for the golden yellow-orange color of saffron; picrocrocin gives bitter flavor, and safranal is responsible for the characteristic aroma of saffron. Saffron is current- ly known as a flavoring agent and a potent natural agent with many health advantages (Azami et al., 2021; Basker & Negbi, 1983; Bolhassani et al., 2014; Mir et al., 2022b). The potential of saffron and its constituents to protect against natural and artificial poisons has enhanced its significance. Due to the high price of saffron and its great demand in the pharmaceutical industry, illegal traffick- ing and adulteration are prevalent nowadays (Alonso et al., 1998). The common adulterants used in saffron in- clude maize silk, marigold floret, horsehair, wool, saffron stamens, red dried silk fiber, and safflower (Carthamus tinctorium L.). Mixing low-grade saffron with high-grade saffron or old stored saffron that has lost its quality with freshly harvested saffron is also a common method of adulteration in saffron (Kumari et al., 2021; Lowell, 1964; Marieschi et al., 2012; Sereshti et al., 2018). The sale and mixing of high-grade Kashmiri saffron with lower-cost Iranian imports is common in India; the resulting mixes are then sold as pure Kashmiri saffron. This trend has deprived saffron cultivators of Kashmir of a significant portion of their revenue (Hussain, 2005). Dyes such as erythrosine, tartrazine, amaranth, sunset yellow, carmoi- sine, picric acid, ponceau S, methyl orange, and Sudan red are also used as adulterants in saffron (Lozano et al., 1999; Patel et al., 2019; Petrakis et al., 2015). Internationally, the grading of saffron is based on the standards formulated by the International Organi- zation for Standardization (ISO). The ISO (ISO 3632- 1:2011) certification ensures customers that the saffron they purchase is authentic and safe to consume. ISO 3632 has classified saffron into three grades (Grade I, II and III) based on the concentration of crocin, picrocrocin and safranal present in saffron [Table 1]. A greater con- centration of these chemicals indicates a better quality of saffron. A quartz cell with a 1 cm pathway is used to measure E1% at 440, 330, and 257 nm wavelengths. The results are obtained by measuring the absorption at three wavelengths using the equation; E1 % 1 cm = (A × 10000]/ (M ×  (100-H)], where E1%  is the specific extinction co- efficient, 1  cm is the path length, A is the absorbance, M is the mass in grams of the saffron sample, H is the moisture and volatile sample material. The moisture and volatile content of the saffron is determined after drying the samples and represented as a mass fraction using the formula: [(initial mass-constant mass)/initial mass] × 100 (Hadizadeh et al., 2007; ISO - 3632-1:2011). Despite international standards, various methods have been reported to detect adulteration and determine the quality of saffron viz UV-Vis spectroscopy (Zalacain et al., 2005; Zougagh et al., 2005) HPLC (Haghighi et al., 2007; Hajimahmoodi et al., 2013; Lozano et al., 1999), mi- cellar liquid chromatography, FTIR (Karimi et al., 2016; Ordoudi et al., 2018), H-NMR (Petrakis et al., 2015), gel-electrophoresis (Paredi et al., 2016). Several factors, including geographical conditions, harvesting period, drying procedure employed, temperature and oxygen ex- posure during storage and adulteration, all have a signifi- cant impact on the quality of saffron (Caballero-Ortega et al., 2004). The primary objective of this research was to estimate the quality range and apocarotenoid content of commercial saffron in Kashmir using UV-Vis spectro- photometry and HPLC analysis. 2 MATERIAL AND METHODOLOGY Saffron in India is cultivated and commercialized in Kashmir. The main local commercial zones of saffron in Kashmir are Srinagar, Pampore and Budgam. Besides local markets, the saffron in Kashmir is also commercial- ized by government-operated commercial emporiums (e.g., Government Kashmir Art Emporiums). Twenty- four samples of saffron were collected from Kashmir, among which six samples were collected from Govern- ment operated commercial emporiums (KAE), six sam- Acta agriculturae Slovenica, 118/3 – 2022 3 Quantification of apocarotenoids in commercial Indian (Kashmiri) saffron using UV-Vis spectroscopy and HPLC analysis ples were collected from the local market of Srinagar (SXR), six samples were collected from Pampore (PAM) district, and six samples were collected from Budgam (BUD). The samples collected were supposed to be yield- ed from the crop year 2019 and processed in 2020 as per their packing. Besides these samples, four samples were collected from Afghanistan (AFG), and two samples were collected from Iran (IRN). The samples collected from KAE, AFG and IRN had an origin certificate and were assured free from any adulteration. One sample was col- lected from Sigma Aldrich (SIG). A total of 31 samples were used for this study. Crocin and safranal standards were purchased from Sigma Aldrich. Picrocrocin was ob- tained from BioMall. HPLC-grade reagents (methanol, acetonitrile) were obtained from Loba Chemie. 2.1 DETERMINATION OF FLORAL WASTE CON- TENT About 1 g of each sample was taken, and each fila- ment was spread on the paper. With the help of forceps, different floral waste components were separated, and the samples were weighed again. The floral waste was taken in shoe glass and weighed. The floral waste content of the sample (wF) was expressed as per ISO guidelines as a percentage by mass, using the relation: wF = (m2 – m1) x 100/mo % Where m0 is the mass, in grams, of the test portion; m1 is the mass, in grams, of the shoe glass; m2 is the mass, in grams, of the shoe glass containing the floral waste. 2.2 DETERMINATION OF MOISTURE AND VOLATILE CONTENT The samples collected needed to be examined for their authenticity. For such purposes, ISO 3632 has pro- vided guidelines for conducting UV-Vis spectroscopy. To calculate E1 %, first, the moisture content of all the sam- ples was calculated. One gram of saffron from each sam- ple was placed in a Petri dish and kept in the oven for 18 hours at 70 0C. After that, samples were weighed again to measure the moisture and volatile matter content (wMV) and is expressed as: wMV = (m0 – m1) x 100/m0 % where m0 is the mass, in grams, of the test portion; m1 is the mass, in grams, of the dry residue. 2.3 UV-VIS SPECTROSCOPY The UV-Vis spectroscopy for samples was per- formed according to ISO guidelines with slight modifi- cations in order to get a greater yield of apocarotenoid compounds. Briefly, 100 mg mass of dried saffron sam- ples was extracted with 5 ml cold 50 % (v/v) ethanol in mortar and pastel. The extract was then transferred to a screw-capped 50 ml tube, and a total amount of 20 ml 50 % (v/v) ethanol was added. Tubes were sonicated for 20 minutes on ice, centrifuged for 15 minutes at 4000 rpm, and washed twice with 5 ml of 50 % (v/v) ethanol. Spectrophotometric technique was employed to analyze the supernatant. For analysis, the supernatant (1 ml) was diluted to 5 ml with 50 percent (v/v) ethanol. The ab- sorption of crocin, safranal, and picrocrocin at 440 nm, 330 nm, and 257 nm, respectively, was used to create a standard curve. The sample supernatants were diluted 100 times, and direct absorbance readings were obtained using a Shimadzu spectrophotometer (1  cm pathway quartz cell) at 440 nm, 330 nm, and 257 nm, respectively. A UV-Vis scan was also obtained to observe peaks in samples of different geographical locations. The results obtained were used to measure E1 % of aqueous saffron extract using the following relation: E1% 1 cm = (A × 10000/ [(M × (100-H)] 2.4 HPLC ANALYSIS For HPLC analysis, 50 mg of powdered saffron sam- ples were extracted with 10 ml of 50 % methanol-water (v/v) and magnetically stirred for 24 hours at 4 °C in the Component λmax Category I Category II Category III Crocin 440 nm ˃ 200 170-200 120-170 Safranal 257 nm 20-50 20-50 20-50 Picrocrocin 330 nm > 70 55-70 40-55 Moisture and volatile matter % (m/m), - 10 10-12 10-12 Table 1: Grades of saffron based on ISO 3632-1:2011 Acta agriculturae Slovenica, 118/3 – 20224 T. G. MIR et al. dark. The samples were then centrifuged for 30 minutes at 5000 rpm. The supernatant was collected and filtered through 0.2 µm syringe filters. For quantitative analysis of crocin, picrocrocin and safranal, 1 ml of 2-nitroaniline was added as an internal standard to each sample before analysis (Caballero-Ortega et al., 2007). The analysis was carried out in a Shimadzu HPLC equipped with quater- nary pumps; coupled to a photo-diode-array detector. Ethanol (50 %, v/v) and acetonitrile (15 %, v/v) were used as the mobile phase. Detection was carried out with an injection volume of 20 μl, a flow rate of 1 ml min−1 with 35-40 min of run time. Crocin was detected at 440 nm, picrocrocin at 250 nm and safranal at 330 nm. A calibra- tion curve was constructed for internal standard  using concentrations of 0.125, 0.25, 0.5, and 1.0 mg ml-1. Quan- titative analysis was carried out in accordance with the molecular absorption coefficient of each peak obtained at the wavelength of maximum absorbance of the compo- nents.  The R2 values ranged from 0.9722to 0.9890, and results were expressed in milligrams per gram of saffron stigmas. 2.5 STATISTICAL ANALYSIS One-way ANOVA was used to compare means and Duncan’s Multiple Range Test (DMRT) was used to assess significance using IBM SPSS (version-20). Two tailored Pearson correlations between apocarotenoid levels with floral waste content and moisture levels were done using IBM SPSS (v. 20). The results were also analyzed using the multivariate analysis technique principal component analysis (PCA) using Origin-2021b (version-9.8b). PCA is a dimensionality-reduction technique often used to decrease the dimensionality of big data sets by convert- ing a large collection of variables into a smaller one that still retains most of the information in the large set. 3 RESULTS AND DISCUSSION The determination of floral waste in the samples was performed by physical separation of floral waste and then measuring its weight. The floral waste in the samples var- ied in range, with samples obtained from SXR and BUD showing a high range of floral waste. KAE samples and sigma samples showed the lowest range of floral waste, while IRN and AFG samples showed a medium to low range of floral waste. Floral waste in the Sigma sample was not detected (Table 2). The moisture/volatile matter content was performed to analyze if the samples had been properly dried and processed. The average moisture level in KAE samples was found to be 6.26  %. Samples from SXR, BUD and PAM showed high levels of moisture and volatile content matter (12.45 %, 7.90 %, and 7.26 %, respectively). The average moisture and volatile content in the AFG and IRN samples was 6.35 % and 542 %, respectively, while in the SIG sample, it was found to be 4.49 % (Table 2). Apocarotenoid content (E1 %) was determined us- ing UV-Vis spectrophotometry. The main objective of this measure analysis was to analyze the quality range of commercial saffron sold in Kashmir. The saffron samples were evaluated in accordance with the ISO 3632-2:2010 guidelines. One-way ANOVA and DMRT were used to compare means and assess the level of significance. The results showed significant variation in all the sam- ples (Table 2). Results showed that average crocin con- tent varied from 198.5 in KAE samples, 135.16 in SXR samples, 184.5 in PAM samples, 166 in BUD samples, 197.25 in AFG samples, 200.5 in IRN samples and 203 in sigma samples. Based on crocin content, it was found that among thirty-one samples, fourteen samples fell in category I, nine fell into category II, five fell in category III and three were counterfeit or adulterated samples as they showed E1 % less than 110. Similarly, picrocrocin expressed as direct reading of the absorbance at 257 nm showed an average concentration of 36.5 in KAE sam- ples, 23.83 in SXR, 33 in PAM, 28.16 in BUD, 34 in AFG, 38.5 in IRN and 32 in SIG sample (Table 3). The safranal content in twenty-nine samples was found to be above 20, thus falling in the optimum range under ISO criteria. Three samples resulted in a safranal content range below 20, which is not optimal as per ISO guidelines. The floral waste and moisture/volatile content in saffron samples were negatively correlated with crocin content values (-0.87, -0.81, respectively). The results were analyzed us- ing PCA analysis. PC1 (76.23 %) and PC2 (18.06 %) ac- counted for 94.29 % of the total variance of the data. The coefficient for both the principal components is given in Table 5. A biplot of samples was obtained to distinguish between adulterated and pure saffron (Figure 1). HPLC analysis provides quick and simple measure- ment of the three major saffron components, with excel- lent linearity, selectivity, sensitivity, and accuracy. The crocetin, picrocrocin, and safranal were determined by HPLC at three wavelengths 440, 250, and 330 nm, re- spectively. The results were analyzed by one-way ANO- VA to compare means, and Duncan’s Multiple Range Test (DMRT) was used to assess significance. The con- centration of these metabolites varied significantly (Table 2). The variations may be attributable to the geographi- cal origin of samples, different drying procedures, stor- age conditions and adulteration (Biancolillo et al., 2020; Delgado et al., 2005; Maghsoodi et al., 2012). The aver- age concentration of crocin varied from 40.64 mg g-1 in Acta agriculturae Slovenica, 118/3 – 2022 5 Quantification of apocarotenoids in commercial Indian (Kashmiri) saffron using UV-Vis spectroscopy and HPLC analysis Sample wF % wMV % UV-Vis Analysis HPLC analysis Crocin (E1 %) 440 nm Picrocrocin (E1 %) 257 nm Safranal (E1 %) 330 nm Crocin (mg g-1) Picrocrocin (mg g-1) Safranal (mg g-1 ) KAE1 0.77 5.26 205a 85a 42a 39.32a 5.36a 0.26bc KAE2 0.41 6.10 212a 86a 44a 43.51a 5.89a 0.31a KAE3 0.66 5.15 178a 62b 28c 45.36a 6.21a 0.26bc KAE4 2.73 7.09 203a 74a 35a 39.95a 6.01a 0.29a KAE5 1.43 6.43 188a 69b 31ab 42.29a 4.03b 0.3a KAE6 6.17 7.53 205a 79a 39a 33.43ab 4.91a 0.27abc SXR1 11.51 9.34 134b 56c 24c 38.49a 4.02b 0.28ab SXR2 4.93 6.73 179a 59c 27c 26.36b 3.16c 0.17c SXR3 5.61 10.3 184a 62b 28c 32.41ab 3.98b 0.2c SXR4 20.49 14.68 80b 43c 23c ND ND 0.18c SXR5 9.49 11.35 162b 57c 25c 34.24ab 3.23c 0.29a SXR6 25.3 22.35 72b 40c 16c 18.26b 2.79c 0.23c PAM1 4.53 12.08 195a 69b 32ab 33.32ab 3.63b 0.29a PAM2 5.68 7.84 152b 65b 29b 38.43a 3.23c 0.26bc PAM3 7.85 7.63 201a 74a 34bc 42.45a 4.02b 0.28ab PAM4 0.60 6.26 163b 72ab 33bc 30.44ab 3.62bc 0.32a PAM5 1.93 8.23 189a 67b 30b 29.4ab 3.14c 0.26bc PAM6 6.25 5.39 207a 82a 40a 33.31ab 4.11b 0.27abc BUD1 2.48 6.19 202a 79a 38a 32.43ab 3.89b 0.29a BUD2 2.80 7.26 185a 60c 29b 26.38b 3.77b 0.27abc BUD3 3.91 8.15 149b 56c 25c 27.39b 3.56c 0.28ab BUD4 16.60 10.28 105b 53c 24c 20.2b 3.04c 0.21c BUD5 11.29 5.33 181a 59c 27c 30.3ab 3.69b 0.23c BUD6 10.24 6.35 176a 58c 26c 32.41ab 3.51c 0.25bc AFG1 1.56 5.68 201a 78a 37a 41.34a 5.1a 0.27abc AFG2 1.34 5.34 208a 81a 39a 30.49ab 4.25b 0.3a AFG3 1.97 6.63 192a 68b 31b 31.42ab 4.07b 0.29a AFG4 3.81 7.76 188a 64b 29bc 38.38a 3.81b 0.25bc IRN1 2.10 5.26 206a 84a 41a 35.42ab 5.33a 0.26bc IRN2 1.92 5.59 195a 75a 36a 35.12ab 5.1a 0.29a SIGMA ND* 4.49 203a 72ab 32ab 34.41ab 4.46ab 0.31a Table 2: Floral waste percentage (wF %), moisture and volatile percentage (wMV %), UV-Vis analysis, HPLC analysis of saffron samples Means followed by the same letter within the columns are not significantly different (p < 0.05) using DMRT *ND- Not Detected KAE samples, 29.952 mg/g in SXR samples, 34.55mg/g in PAM samples, 28.18 mg/g in BUD samples, 35.40 mg g-1 in AFG samples, 35.27 mg g-1 in IRN samples and 34.41 mg g-1 in sigma sample. Safranal, one of the main components responsible for the fragrance of the spice, is soluble in polar solvents and poorly soluble in nonpolar solvents. The safranal content as per the ISO 3632 (2011) method cannot be categorized in any grade as the ISO Acta agriculturae Slovenica, 118/3 – 20226 T. G. MIR et al. Sample Origin ISO Category Crocin (E1 %) 440 nm Safranal (E1 %) 257 nm Picrocrocin (E1 %) 257 nm KAE I (4) 203-212 28-31 62-69 II (2) 168-178 35-42 74-84 SXR II (2) 179-184 27-28 59-62 III (2) 134-162 24-25 56-57 IV* (2) 72-80 16-23 40-43 PAM I (3) 195-207 32-40 64-82 II (1) 189 30 67 III (2) 152-163 29-33 65-73 BUD I (1) 202 38 79 II (3) 176-185 26-29 58-60 III (1) 149 25 56 IV* (1) 105 24 53 AFG I (3) 192-208 31-39 68-81 II (1) 188 29 64 IRN I (2) 36-41 75-24 75-24 SIG I (1) 203 32 72 Table 3: Quality characteristics of saffron obtained from different geographical locations using the ISO-3632 method *Highly adulterated or counterfeit saffron samples Figure 1: PCA analysis (biplot) of saffron samples (UV-Vis analysis) Acta agriculturae Slovenica, 118/3 – 2022 7 Quantification of apocarotenoids in commercial Indian (Kashmiri) saffron using UV-Vis spectroscopy and HPLC analysis Figure 2: PCA analysis (biplot) of saffron samples (HPLC analysis) method doesn’t provide a precise classification of grades of saffron based on safranal content. The average safranal content differed significantly across KAE (0.28 mg g-1 ), SXR (0.22 mg g-1 ), PAM (0.28 mg g-1 ), BUD (0.25 mg g-1 ), AFG (0.27 mg g-1 ), IRN (0.27 mg g-1 ), and sigma samples (0.31 mg g-1 ). Meanwhile, average picrocrocin content ranged from 5.40 mg g-1 in KAE, 3.43 mg g-1 in SXR, 3.62 mg g-1 in PAM, and 3.57 mg g-1 in BUD sam- ples, 4.30 mg g-1 in AFG, 5.21 mg g-1 in IRN and 4.46 mg g-1 in SIG sample (Table 4). Crocin and picrocrocin were not detected in SXR4 sample. A biplot of samples was obtained to analyze the relation between metabolites in samples using PCA (Figure 2). PC1 (92.31 %) and PC2 (7.16 %) accounted for 99.47 % of the total variance of the data. The coefficient for both the principal compo- nents is given in Table 5. The results obtained from UV-Vis spectroscopy showed that saffron samples from KAE were of the high- est grade compared to other saffron obtained from other commercial sites. The saffron from PAM and BUD com- mercial sites showed a moderate range of quality. The samples from AFG and IRN fell in grade I and II as per ISO parameters. The saffron from SXR markets showed the lowest grade compared to other samples. The HPLC analysis showed a higher concentration of apocarotenoid content in KAE samples, followed by AFG and IRN sam- ples. The SXR samples showed the lowest quality and apocarotenoid content, which indicates an indication of adulteration. 4 CONCLUSION The evaluation of the quality of saffron selections was done by UV-Vis Spectroscopy according to the limit set by the ISO 3632, and the determination of apocarot- enoid content was analyzed by HPLC analysis. The UV- Vis spectrophotometric results categorized the sample into different grades as per standards formulated by ISO. Only fourteen samples were identified as Grade I, and 13 samples were either grade II or grade III. The remain- ing 3 samples were found to be highly adulterated. The results obtained from HPLC analysis showed significant variation. The highest concentration of apocarotenoids Acta agriculturae Slovenica, 118/3 – 20228 T. G. MIR et al. Sample Origin Crocin (mg g-1 ) Safranal (mg g-1 ) Picrocrocin (mg g-1 ) KAE 33.43-45.36 0.26-0.31 4.03-6.21 SXR 18.26-38.49 0.17-0.29 2.79-4.02 PAM 29.40-42.45 0.26-0.32 3.14-4.11 BUD 20.20-32.43 0.21-0.29 3.04-3.89 AFG 30.49-41.34 0.25-0.30 3.81-5.10 IRN 35.12-35.42 0.26-0.29 5.10-5.33 SIG 34.41 0.31 4.46 Table 4: HPLC-based concentration range of crocin, safranal and picrocrocin of saffron samples obtained from different geographical locations Variable Coefficients of PC1 Coefficients of PC2 UV-Vis analysis Crocin 0.5555 0.82244 Safranal 0.58313 -0.49034 Picrocrocin 0.59278 -0.28836 HPLC analysis Crocin 0.61407 -0.28918 Safranal 0.51423 0.85245 Picrocrocin 0.59875 -0.43554 Table 5: Loading of the first two principal components (PC’s) for concentration of metabolites was found in KAE samples, followed by AFG, IRN, PAM and BUD samples. Samples from SXR showed the least concentration of apocarotenoids, indicating a high level of adulteration. 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